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PubMed2005

Whole genome SNP arrays using DNA derived from formalin-fixed, paraffin-embedded ovarian tumor tissue.

Array-based genotyping platforms, such as the Affymetrix mapping array, have been validated as reliable methods for obtaining high-resolution copy number and allele status information when using DNA derived from fresh tissue sources. However, the suitability of such systems for the examination of DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissues has not been tested. Therefore, we analyzed DNA derived from five matching fresh frozen and FFPE ovarian tumors for gene copy number changes and loss of heterozygosity using the Affymetrix GeneChip Human Mapping 10 K Array Xba 131. The data was analyzed using Affymetrix proprietary software, GeneChip DNA Analysis Software, and Chromosome Copy Number Tool. The average SNP call rate (rate of successful genotype identification) of the fresh samples was 89.44% (range 78.72-96.22%, median 92.72%) and was only slightly lower for the matching FFPE samples at 83.48% (range 76.93-93.17%, median 82.60%). The average concordance (rate of agreement between successful genotype calls) between the fresh and matching FFPE samples was 97.06% (range 92.70-99.41%, median 97.52%). Loss of heterozygosity (LOH) profiles of the fresh and FFPE samples were essentially identical across all chromosomes. Copy number data was also comparable, although the quantification of copy number changes appears overstated in the FFPE samples. In conclusion, we have shown that it is possible to achieve high-performance outcomes using FFPE-derived DNA in the Affymetrix 10 K mapping array. This advance will open up vast archival tissue resources to high-resolution genetic analysis and unlock a wealth of biological information.

DNA↗

PubMed2005

Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia.

To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2%) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20%) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.

Amino Acid Sequence↗

PubMed2001

Comparison of endogenous feline leukemia virus RNA content in feline vaccine and nonvaccine site-associated sarcomas.

OBJECTIVE: To determine whether feline vaccine site-associated sarcomas (VSS) contain a higher amount of endogenous FeLV (enFeLV) RNA, compared with feline nonvaccine site-associated sarcomas (non-VSS). SAMPLE POPULATION: Formalin-fixed paraffin-embedded (FFPE) tissues from 50 VSS and 50 cutaneous non-VSS. PROCEDURE: RNA was extracted from FFPE sections of each tumor, and regions of the long terminal repeat (LTR) and envelope (env) gene of enFeLV were amplified by use of reverse transcriptase-polymerase chain reaction (RT-PCR). The density of each RT-PCR product band for enFeLV was compared with that of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An integrated density value (IDV) was determined by use of densitometry, and the IDV ratio for enFeLV to GAPDH was calculated for each enFeLV primer set. RESULTS: The median (interquartile range) of the IDV ratio for the enFeLV LTR primer set was 0.52 (0.26 to 1.17) for the VSS group and 0.84 (0.21 to 1.53) for the non-VSS group. The median (interquartile range) of the IDV ratio for the enFeLV env primer set was 0.60 (0.37 to 0.91) for the VSS group and 0.59 (0.36 to 1.09) for the non-VSS group. CONCLUSIONS: Because the amount of enFeLV RNA within the LTR and env gene was not significantly different between the VSS and non-VSS groups, enFeLV replication or expression is unlikely to be involved in VSS development.

Animals↗

PubMed2006

Real-time quantitative RT-PCR shows variable, assay-dependent sensitivity to formalin fixation: implications for direct comparison of transcript levels in paraffin-embedded tissues.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is a versatile tool for precise quantification of gene expression. Formalin-fixed and paraffin-embedded (FFPE) tissue is well suited for qRT-PCR, if RNA extraction is optimized and small amplicon sizes are used. However, little is known whether individual assays may show variable sensitivity to fixation. This is of great importance, if a direct comparison of different transcripts is performed within the same sample, such as for mRNA splice variants. We established a cell culture model to test for and quantify differences in performance of individual qRT-PCR assays on FFPE as compared with fresh material, using TaqMan methodology. RNA was isolated from 7 different cell lines either directly or after preparation of a FFPE cell block. RNA from both sources was reverse transcribed and gene expression quantified using 13 different TaqMan assays. All assays allowed highly reproducible target quantification, using both fresh and FFPE-derived cDNA. The 13 assays showed an average Ct difference of 3.2 between fresh and FFPE cells, if identical amounts of cDNA were used as template. However, the Ct shifts varied from 1.8 to 5.1 for individual assays, indicating variable resistance to fixation. These Ct shift differences were statistically highly significant in 27/78 (35%) of all possible combinations of assays. Because the Ct shift remained constant for each assay, they could be used for calculation of correction factors which rendered FFPE-derived expression data highly comparable to those obtained from fresh material, and as a consequence among each other. Thus, a standardized assessment of qRT-PCR assay efficiencies in FFPE allows for precise intraindividual comparison of mRNA species, such as splice variants with different biologic functions, in archival tissues.

Biological Specimen Banks↗

PubMed2005

Protein-embedding technique: a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections.

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a 'block' that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.

Animals↗

PubMed2005

Liquid Tissue: proteomic profiling of formalin-fixed tissues.

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.

Chromatography, Liquid↗

PubMed1996

Multiple drug resistance-related messenger RNA expression in archival formalin-fixed paraffin-embedded human breast tumour tissue.

A method is described by which RNA, suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, can be extracted from formalin-fixed paraffin-embedded (FFPE) tissues and subsequently used for detecting the expression of several genes. Using this technique, RNA can be extracted from specimens, quantified, reverse transcribed and regions of interest amplified and analysed within 36 h. The tissue specimens included in this study were from human breast carcinoma, investigating a range of genes associated with the development and/or maintenance of multiple drug resistance (MDR). This technique, applied to archival tissues, offers great potential for increasing our understanding of alterations in expression levels of genes associated with MDR. The method developed is also applicable to studies on expression of other genes in paraffin-embedded tissues.

Adult↗

PubMed2003

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

DNA Primers↗

PubMed2002

Detection of SYT-SSX fusion transcripts in archival synovial sarcomas by real-time reverse transcriptase-polymerase chain reaction.

Synovial sarcomas comprise approximately 5% of soft tissue sarcomas and occur primarily in young adults. The t(X;18) (p11.2;q11.2) has been demonstrated to be highly characteristic of synovial sarcomas, and the resulting SYT-SSX fusion transcripts have been shown to be useful diagnostic markers. We have developed a real-time, reverse transcriptase-polymerase chain reaction (RT-PCR) multiplex assay for the identification of the primary fusion transcript types (SYT-SSX1 and SYT-SSX2) from formalin-fixed, paraffin-embedded (FFPE) tissues. Twenty-nine of 30 (96.7%) histologically diagnosed FFPE synovial sarcomas were positive for the presence of either the SYT-SSX1 or SYT-SSX2 fusion transcripts. Ten of 16 (62.5%) and five of 16 (31.25%) monophasic fibrous synovial sarcomas were positive for SYT-SSX1 and SYT-SSX2, respectively. One of 16 (6.25%) monophasic fibrous synovial sarcomas was negative for either SYT-SSX fusion transcript. Twelve of 14 (85.7%) and 2 of 14 (14.3%) biphasic synovial sarcomas were positive for SYT-SSX1 and SYT-SSX2, respectively. All 13 non-synovial sarcomas tested were negative for SYT-SSX1 and SYT-SSX2 fusion transcripts. This method is a relatively simple and rapid procedure for the detection of the t(X;18)(p11.2;q11.2).

Biomarkers, Tumor↗

PubMed2005

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA.

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.

DNA, Neoplasm↗

PubMed1996

Hepatitis C and B virus infections in hepatocellular carcinoma. Analysis of direct detection of viral genome in paraffin embedded tissues.

BACKGROUND: Although there have been many seroepidemiologic studies on hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC) occurrence, the actual role of HCV in hepatocarcinogenesis is unknown. METHODS: We have previously reported on a highly sensitive method of detecting and identifying sequences of RNA genome in formalin fixed, paraffin embedded (FFPE) tissue by polymerase chain reaction (PCR) assay. Using this method, we carried out a retrospective study to determine the prevalence of HCV and hepatitis B virus (HBV) genomes in FFPE specimens from 102 Japanese patients with HCC. RESULTS: HCV-RNA was detected by nested PCR reverse transcription (RT) in 64 of the 102 patients (62.7%), and 78.1% (50/64) of those HCV-RNA-positive patients had HCV genotype II. HCV-RNA was present in 54 of 70 (77.1%) anti-HCV-positive patients, and also in 5 of 20 (25%) anti-HCV-negative patients. HBV-DNA was detected by nested PCR in FFPE liver specimens from 21 of 102 (20.6%) patients. HBV-DNA positivity was consistent with seropositivity for serum HBV markers in 17 of these 21 patients (80.9%). HBV-DNA was present in FFPE samples from 2 patients who were seronegative for HBV markers, and in 1 patient who was seropositive for anti-HBs. Double infection of these two viruses was found in 6 patients (5.9%). Three patients (2.9%) were negative for both hepatic viral genomes and serum viral markers. CONCLUSIONS: The precise prevalence of HCV and/or HBV infection among HCC patients can be determined by studying routinely-processed FFPE HCC samples preserved for up to 11 years using the technique of nested PCR. HCV-RNA was detected in the majority of our HCC cases; type II was the most common genotype of HCV encountered. The incidence of HCV-associated HCC was three times greater than that of HBV. Thus, the hepatitis virus infection most frequently associated, and probably ectologically implicated, with HCC in Japanese people is HCV infection.

Adult↗

PubMed2006

A multiplex PCR predictor for aCGH success of FFPE samples.

Formalin-fixed, paraffin-embedded (FFPE) tissue archives are the largest and longest time-spanning collections of patient material in pathology archives. Methods to disclose information with molecular techniques, such as array comparative genomic hybridisation (aCGH) have rapidly developed but are still not optimal. Array comparative genomic hybridisation is one efficient method for finding tumour suppressors and oncogenes in solid tumours, and also for classification of tumours. The fastest way of analysing large numbers of tumours is through the use of archival tissue samples with first, the huge advantage of larger median follow-up time of patients studied and second, the advantage of being able to locate and analyse multiple tumours, even across generations, from related individuals (families). Unfortunately, DNA from archival tissues is not always suitable for molecular analysis due to insufficient quality. Until now, this quality remained undefined. We report the optimisation of a genomic-DNA isolation procedure from FFPE pathology archives in combination with a subsequent multiplex PCR-based quality-control that simply identified all samples refractory to further DNA-based analyses.

Breast Neoplasms↗

PubMed2001

Automated immunohistochemical staining of formalin-fixed and paraffin-embedded tissues using a catalyzed signal amplification method.

An immunohistochemical assay using catalyzed signal amplification (CSA), which is based on the peroxidase catalyzed deposition of biotinylated tyramide, is a highly sensitive method to visualize weak immunohistochemical signals originating from rare antigens or masked antigens in formalin-fixed, paraffin-embedded (FFPE) tissues. However, CSA methods are hampered by poor reproducibility and the complexity of their staining procedures. In this study, we aimed to apply the CSA procedure to a capillary gap-based, automated immunostainer, TechMate Horizon, to perform immunohistochemical signal amplification effectively and reproducibly. A variety of cellular antigens previously considered to be undetectable in FFPE human specimens were selected and examined with the automated immunostainer. Compared with the manual CSA staining method that takes more than 2 hours, the automated CSA method took less than 2 hours to complete. The staining results from the automated CSA method presented higher reproducibility, as well as lower background owing to well-regulated, punctual staining and washing at every step of the procedure. Conclusively, the automation of the CSA method enabled us to perform the time-consuming and complicated CSA amplification technique with minimal effort in an accurate, consistent, and reproducible manner.

Antigens↗

PubMed2000

Myogenin is a specific marker for rhabdomyosarcoma: an immunohistochemical study in paraffin-embedded tissues.

Myogenin belongs to a group of myogenic regulatory proteins whose expression determines commitment and differentiation of primitive mesenchymal cells into skeletal muscle. The expression of myogenin has been demonstrated to be extremely specific for rhabdomyoblastic differentiation, which makes it a useful marker in the differential diagnosis of rhabdomyosarcomas (RMS) from other malignant small round cell tumors of childhood. Commercially available antibodies capable of detecting myogenin in routinely processed formalin-fixed paraffin-embedded (FFPE) tissue are now available. In this study, we evaluated myogenin expression using the monoclonal myf-4 antibody (Novocastra Labs) on FFPE in a large number of pediatric tumors in order to define the clinical utility of this marker. A total of 119 tumors were studied. These included 48 alveolar RMS (ARMS), 20 embryonal RMS (ERMS), one spindle cell RMS, 16 Ewing's sarcomas (ES), six nephroblastomas, two ectomesenchymomas, seven precursor hematopoietic neoplasms, five olfactory neuroblastomas, three neuroblastomas, six desmoplastic small round cell tumors, and five rhabdoid tumors. Distinct nuclear staining for myogenin was noted in all 69 RMS. Notably, the number of positive tumor cells differed between the ARMS and ERMS. In ARMS, the majority of tumor cells (75 to 100%) were positive, in contrast to ERMS, in which the positivity ranged from rare + to 25% in all but three tumors. Additionally, myogenin positivity was seen in two of two ectomesenchymomas and in two nephroblastomas with myogenous differentiation. All other tumors were clearly negative. Our results indicate that staining for myogenin is an extremely reliable and specific marker for rhabdomyoblastic differentiation. It gives consistent and easily interpretable results in routinely fixed tissues.

Biomarkers, Tumor↗

PubMed2004

Detection of hepatitis C virus RNA in formalin-fixed paraffin-embedded sections with digoxigenin-labeled cRNA probes.

Although recent studies have analyzed Hepatitis C (HCV) infections in liver tissue by in situ hybridization (ISH), many of these studies have been of limited diagnostic utility because of the low copy numbers of HCV in formalin-fixed paraffin-embedded (FFPE) tissue and failure to correlate the ISH analysis with other methods of detecting HCV. Thirty six cases of liver biopsies from patients with known HCV antibody status including 20 cases of serum HCV positive and 16 cases of serum HCV negative were analyzed. All cases showed histologic features suggestion of HCV infection. Analyses of all 36 cases were done by RT-PCR combined with Southern hybridization (RT-PCR-SH) and in situ hybridization (ISH). A prolactin riboprobe was used as a negative control. Immunohistochemistry (IHC) with an antibody against HCV (Rb 246) was also used to analyze HCV viral protein in the tissues. Of the 20 serum antibody-positive cases, RT-PCR-SH detected 18 positive cases, while ISH and IHC detected 19 and 16 positive cases, respectively. Of the 16 serum antibody-negative cases, RT-PCR-SH detected 8 positive cases while ISH and IHC detected 8 and 11 positive cases, respectively. A positive ISH signal for HCV was also detected in some lymphocytes and bile ducts in the liver. These results show that ISH with a highly specific riboprobe is comparable to RT-PCR-SH for detection of HCV infection in liver tissue.

Biopsy↗

PubMed1997

Polymerase chain reaction based gene rearrangement studies in the diagnosis of follicular lymphoma--performance in formaldehyde-fixed tissue and application in clinical problem cases.

The diagnostic potential of PCR-based studies on the immunoglobulin heavy chain (IgH) gene rearrangements and the t(14;18) translocation was evaluated in a series of 30 benign and 46 malignant follicular lymphoid lesions including difficult problem cases using formaldehyde-fixed and paraffin-embedded (FFPE) tissue. Clonal IgH gene rearrangement was documented by PCR in 38/46 follicular lymphomas using two alternative (FR3A and FR2A-based) primer systems: all benign hyperplasias showed a polyclonal pattern. The t(14;18) translocation was documented in 20/46 cases, including cases that did not show a clonal IgH gene amplification; a weak signal was seen in 6 benign hyperplasias probably related to the recently described rare non-neoplastic cells carrying the t(14;18) translocation. Of the translocations in follicular lymphomas, 18 involved the major breakpoint region (MBR) and two the minor cluster region (MCR). The diagnosis of follicular lymphoma could be reached with at least one marker in over 90% of the cases. With these tests we diagnosed an incidental follicular lymphoma in the axillary dissection for breast carcinoma, and confirmed benign nature of an extreme follicular hyperplasia with a 2200 g spleen. One diffuse large cleaved cell lymphoma with subsequent follicular small cleaved cell lymphoma was diagnosed to have a similar t(14;18) translocation showing a link between the two seemingly different lymphomas.

Adult↗

PubMed2026

Determinants of nucleic acid quality in formalin-fixed paraffin-embedded bladder urothelial carcinoma specimens: effects of specimen type, fixation conditions, and storage time.

Formalin-fixed paraffin-embedded (FFPE) tissue is the principal substrate for cancer genomic profiling, yet pre-analytical factors affect nucleic acid quality. In our workflow, transurethral resection (TUR) specimens of bladder urothelial carcinoma are fixed promptly but exposed to electrocautery, whereas robot-assisted radical cystectomy specimens are refrigerated overnight before fixation; we compared nucleic acid quality between these specimen types. We analyzed 56 FFPE primary bladder urothelial carcinoma specimens (33 TUR, 23 cystectomy). DNA quality was assessed by the DNA Integrity Number (DIN) and short-to-long cycle threshold ratio (S/L Ct ratio), and RNA quality by the RNA Integrity Number (RIN) and percentage of RNA fragments ≥200 nucleotides (DV200), with group comparisons, Spearman correlations, and multivariable regression (specimen type, fixation duration, storage time). The median DIN (4.4 vs. 3.8, p = 0.004) and S/L Ct ratio (0.943 vs. 0.894, p < 0.001) were higher in TUR than cystectomy specimens; the RIN was also higher (1.9 vs. 1.7, p = 0.040), though low in both groups, and DV200 did not differ (50.3% vs. 47.7%, p = 0.934). In multivariable analysis, TUR specimen type independently predicted higher DIN (β = +0.460, p = 0.040) and S/L Ct ratio (β = +0.442, p = 0.009), whereas longer storage time independently decreased the S/L Ct ratio (β = -0.513, p < 0.001) and RIN (β = -0.352, p = 0.010). In our institutional workflow, TUR specimens showed higher DNA quality metrics than robot-assisted radical cystectomy specimens subjected to overnight refrigerated pre-fixation delay, whereas this workflow-associated advantage did not clearly extend to DV200-defined RNA fragment-length quality. Storage time was associated with selected aspects of nucleic acid deterioration.

Humans↗

PubMed2006

Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase.

BACKGROUND: In patients with cancer, one of the main mechanism of resistance to antimetabolite drugs is related to higher levels of thymidylate synthase (TS) activity. METHODS: To investigate the association between TS expression and histopathologic data, 56 resection specimens from patients with nonsmall cell lung carcinoma (NSCLC) were collected consecutively. TS messenger RNA (mRNA) was evaluated in tumor specimens by using real-time polymerase chain reaction (PCR) analysis; protein expression was evaluated by using immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) specimens; and the analysis of TS transcriptional regulation activity was performed by using real-time PCR analysis in snap-frozen normal and tumor specimens. RESULTS: The amplification of the TS gene from FFPE tissues was obtained from all samples, with a median level (unit-less ratio) of 1.45 (range, 0.34-5.24); whereas positive TS status at IHC (>10% positive cells) was detected in 56% of samples. It is noteworthy that TS expression was significantly higher in squamous cell carcinoma compared with adenocarcinoma when both mRNA levels (2.17 vs. 1.16; P < .0001) and protein levels (P = .0269) were considered in FFPE specimens, and a strong association was observed between mRNA and protein expression (P = .00017). Moreover, higher TS levels were observed in high-grade tumors (P = .0389 and P = .0068 for mRNA and protein quantification, respectively). The analysis in snap-frozen samples revealed that the TS gene was up-regulated strongly in tumors (P = 3.8 x 10(-12)), and an 8-fold increase (as a cut-off value) in the TS mRNA ratio between tumor and corresponding normal tissue was detected in 32 of 56 patients (57%) bearing preferentially squamous cell tumors (P = .0022) and high-grade tumors (P < .001). CONCLUSIONS: Data from the current study consistently indicated higher TS expression levels in squamous cell and in high-grade carcinomas. This information may be useful in selecting which patients with NSCLC should receive treatment with TS-inhibiting agents.

Adult↗