Heat-induced Antigen Retrieval Research Articles

Abstract C11: Protein biomarkers predictive of outcome in EGFR inhibitor-treated non-small cell lung cancer patients with EGFR wildtype

Abstract Background: EGFR inhibitors, erlotinib and gefitinib, cause tumor shrinkage in approximately 70% of non-small cell lung cancer (NSCLC) patients with activating mutations in EGFR, but may also be efficient in a subset of patients with EGFR wildtype, as indicated by a retrospective analysis of the BR.21 trial. However, there is currently a lack of biomarkers predictive for outcome of EGFR inhibitor-treated NSCLC patients with EGFR wildtype. The identification of such biomarkers was the aim of this study. Materials and methods: Formalin-fixed paraffin-embedded tissue blocks from 50 NSCLC patients with EGFR wildtype, who received treatment with erlotinib/gefitinib were included and stratified according to progression-free-survival (PFS). Twelve patients experienced no progression for >6 months (Good Response Group), while 38 patients experienced progression in <6 months (Poor Response Group). From formalin-fixed paraffin-embedded surgical resections, areas of high tumor content were isolated; peptides were extracted in triethyl ammonium bicarbonate/acetonitrile buffer by heat-induced antigen retrieval, and then fractionated by hydrophilic-interaction liquid chromatography (HILIC) followed by quantification and identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were analyzed in technical duplicates, and only proteins identified with minimum 2 unique peptides were used for further analysis. Results: The patient cohort represented 20 men and 30 women. Two were non-smokers, 45 were smokers/former smokers and three were unknown. 42 exhibited adenocarcinoma histology and eight exhibited bronchioalveolar carcinoma histology. The majority of patients were treated with erlotinib (n = 47), while three received gefitinib. Mean PFS of patients in the Good Response Group was 455 days, while patients in the Poor Response Group had a mean PFS of 66 days, indicating that there are indeed patients with EGFR wildtype who benefit from EGFR inhibitors. From the quantitative LC-MS/MS analysis, we identified and quantified 3152 high-confident proteins across the 50 patients with ≥2 unique peptides. Softwares R and GProX were used for statistical analysis. Data was quantile normalized and revealed 30 proteins which were found to be differentially expressed between the two groups with a p-value <0.01. Among the most differentially expressed proteins, we found proteins involved with cytoskeletal rearrangement, intracellular transport, protein translation, transcription regulators and kinases. Conclusions: From the PFS analysis of our patient cohort, it is clear that patients exhibiting EGFR wildtype may also benefit from treatment with EGFR inhibitors. Using unbiased mass spectrometry, we identified 30 differentially expressed proteins with the potential to predict outcome of EGFR inhibitor-treated NSCLC patients with EGFR wildtype. Citation Format: Kirstine Jacobsen, Brian Hood, Karen Ege Olsen, Thomas Conrads, Henrik Ditzel. Protein biomarkers predictive of outcome in EGFR inhibitor-treated non-small cell lung cancer patients with EGFR wildtype. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C11.

Prognostic Significance of Nuclear β-Catenin Expression in Patients with Colorectal Cancer from Iran.

Background:Beta catenin plays a key role in cancer tumorigenesis. However, its prognostic significance in patients with colorectal cancer (CRC) remains controversial. It has been demonstrated that 90% of all tumors have a mutation in individual components of multiple oncogenes in Wnt/β-catenin pathway. Accumulation of nuclear β-catenin in cytoplasm leads to uncontrolled cell proliferation. Thus, nuclear β-catenin accumulation may be a valuable biomarker associated with invasion, metastasis and poor prognosis of CRC.Objectives:In this study the prognostic value of beta catenin expression in 165 Iranian CRC patients was evaluated.Patients and Methods:In this cross sectional retrospective study immunohistochemistry analyses of formalin-fixed paraffin-embedded (FFPE) tumor tissues were performed to characterize the expression of nuclear β-catenin in a series of 165 Iranian patients with colorectal carcinoma. Heat-induced antigen retrieval using the microwave method was applied for all staining procedures. Staining was scored independently by two observers, and a high level of concordance (90%) was achieved. Statistical analysis was done using the SPSS software for Windows, version 13.0.0 (SPSS Inc., Chicago, IL). Two-tailed P < 0.05 was considered statistically significant.Results:The patients consisted of 85 males and 80 females. Eighty-eight patients had primary tumor of the rectum and sigmoid, while 77 patients had primary tumor of the colon. The mean period of follow-up was 47.2 ± 10 months and the median period of follow-up was 38 months (range 6 - 58) for each patient. Of 165 tumors, 32 tumors (19.39 %) showed expression of β-catenin and 133 (80.6 %) were negative for β-catenin expression. Based on our findings the distribution of Microsatellite Instability (MSI) status differed between patients with nuclear β-catenin positive and negative tumors and this difference was significant (P = 0.001). Patients with nuclear β-catenin positive expression profile were found to be younger than patients with negative nuclear β-catenin expression (P = 0.010). Univariate and multivariate analysis showed that tumors with β-catenin expression had a poorer prognosis compared to tumors without β-catenin expression.Conclusions:According to our findings, the distribution of nuclear b-catenin expression is a poor prognostic marker in patients with colon cancer.

Identification of 5-Bromo-2’-Deoxyuridine-Labeled Cells during Mouse Spermatogenesis by Heat-Induced Antigen Retrieval in Lectin Staining and Immunohistochemistry

DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2'-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.

Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens: Procedures and Mechanisms.

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

Brief Formalin Fixation and Rapid Tissue Processing Do Not Affect the Sensitivity of ER Immunohistochemistry of Breast Core Biopsies

Recent studies have questioned the supporting evidence for the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines of the 8-hour minimum fixation time required for estrogen receptor immunohistochemistry (ER-IHC) assays in breast cancer. We investigated whether brief formalin fixation together with rapid tissue processing affects the sensitivity of ER in core breast biopsies. Five core samples each from 22 mastectomy specimens were collected and fixed in 10% formalin for periods ranging from 30 minutes to 1 week. Core 5 was fixed and processed according to the ASCO/CAP guidelines. ER-IHC was performed following heat-induced antigen retrieval using antibody 1D5. The proportion and intensity of reaction was recorded using the Q score. Five of 22 cancers were ER negative in all cores. In 17 ER-positive cases, no differences were found in the intensity of reaction between 30 minutes and 1 week of formalin fixation. Similarly, no difference was observed in the Q scores of rapidly and conventionally processed control tumor cores. Brief formalin fixation along with rapid processing has no negative effect on the sensitivity of ER-IHC in breast core biopsies. This combination significantly reduces the turnaround time for preparing breast needle biopsy specimens.

Immunohistochemical staining of influenza virus in tissues.

Immunohistochemical methods are commonly used for studying the pathogenesis of influenza A virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of influenza virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to the virus antigen; antibody-antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric end product. Visualization of influenza virus antigen in tissues is based on chromogen deposition in the nucleus and cytoplasm of infected cells.

Immunoelectron Microscopy with the Post-Embedding Method Based on Heat-Induced Antigen Retrieval: Techniques and Mechanisms

Immunoelectron Microscopy with the Post-Embedding Method Based on Heat-Induced Antigen Retrieval: Techniques and Mechanisms

Evaluation and comparison of expression of p63 in odontogenic keratocyst, solid ameloblastoma and unicystic ameloblastoma.

Background and Objectives:The behavior of odontogenic lesions varies with some tumors behaving like a cyst and some cysts behaving like tumors. p63, a member of the p53 family of tumor suppressor genes has recently come into light in view of its role as an oncogene. The aim of the present study was to investigate the expression of p63 protein in OKC, Solid ameloblastoma, Unicystic Ameloblastoma and Follicular tissue.Materials and Methods:p63 expression was compared in 12 cases of OKC, 12 Solid Ameloblastoma, 14 cases of Unicystic ameloblastoma and 10 cases of Follicular tissue using immunohistochemical technique. All 48 cases were subjected to heat-induced antigen retrieval method using citrate buffer in a pressure cooker. Then the sections were stained with anti-p63 polyclonal antibody and visualized using super sensitive polymer HRP detection system. In each case, number of cells showing p63 positivity were assessed in two compartments - basal and suprabasal and compared.Results:Statistical analysis showed that p63 expression in the suprabasal compartment in Odontogenic keratocysts was equivalent to that of central neoplastic cells of Solid Ameloblastoma and Unicystic Ameloblastoma type 3. Statistically significant difference in the expression of p63 was observed between OKC and Unicystic Ameloblastoma Type 1 and Solid Ameloblastoma and Unicystic Ameloblastoma Type 1.Conclusion:We conclude that the higher expression of p63 in these odontogenic lesions correlates well with their aggressive behavior and thereby suggesting alterations in treatment modalities.

βIII-Tubulin Overexpression Is an Independent Predictor of Prostate Cancer Progression Tightly Linked to ERG Fusion Status and PTEN Deletion

Evidence suggests that class III β-tubulin (βIII-tubulin) may represent a prognostic and predictive molecular marker in prostate cancer. βIII-Tubulin expression was determined by IHC in 8179 prostate cancer specimens in a TMA format. Results were compared with tumor phenotype, biochemical recurrence, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) status, and deletions on PTEN, 3p13, 5q21, and 6q15. βIII-Tubulin expression was detectable in 25.6% of 8179 interpretable cancers. High βIII-tubulin expression was strongly associated with both TMPRSS2:ERG rearrangement and ERG expression (P<0.0001 each). High βIII-tubulin expression was tightly linked to high Gleason grade, advanced pT stage, and early prostate-specific antigen (PSA) recurrence in all cancers (P<0.0001 each), but also in the subgroups of ERG-negative and ERG-positive cancers. When all tumors were analyzed, the prognostic role of βIII-tubulin expression was independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features, such as biopsy specimen Gleason grade, preoperative PSA, cT stage, and βIII-tubulin expression (P<0.0001 each). βIII-Tubulin expression was associated with PTEN (P<0.0001) when all tumors were analyzed, but also in the subgroups of ERG-negative and ERG-positive cancers. βIII-Tubulin expression is an independent prognostic parameter. The significant associations with key genomic alterations of prostate cancer, such as TMPRSS2:ERG fusions and PTEN deletions, suggest interactions with several pivotal pathways involved in prostate cancer.

Abstract P4-07-07: Analysis of miRNAs and proteins relations in breast cancer

Abstract Introduction / Objectives MicroRNAs (miRNAs) constitute a new class of small noncoding RNAs that control post-transcriptionally the expression of gene products, either modulating directly protein translation, or regulating the stability of messenger RNA. There is increasing evidence of the role that miRNAs play in regulating breast cancer gene expression. However, there is little knowledge about the function and targets of miRNAs, and how they regulate complete processes or pathways. The main objective of this study was to unravel biological processes and signaling pathways regulated by miRNAs in breast cancer comparing their expression with the expression of the proteins they regulate. New statistical approaches were conducted in order to associate miRNA and protein quantification results with breast cancer subtype and to evaluate the association of miRNAs and protein expression patterns. Materials and Methods MicroRNA and protein expression were obtained from 79 breast cancer FFPE samples (16 TNBCs and 63 Luminal tumors). RNA was extracted from FFPE samples using RecoverAll (Ambion). MicroRNA expression was analyzed by RT-qPCR using TaqMan Arrays (Applied Biosystems). We selected for subsequent analysis those miRNAs with significant correlation between FF and FFPE samples. Protein extracts from FFPE samples were prepared in 2% SDS buffer using a protocol based on heat-induced antigen retrieval (Gámez-Pozo A et al. Mol Biosyst. 2011; 7: 2368-74). Protein abundance was calculated on the basis of normalized spectral protein intensity (LFQ intensity) using MaxQuant. Probabilistic graphical models are being applied successfully to represent complex biological systems as networks. In this phase of the study, we have chosen an appropriate methodology in the analysis of high dimensional data selecting a forest which minimizes the BIC criterion. This procedure extends the Chow and Liu's approach to the Gaussian case. The software used for implementation is based on the R library gRapHD. Results We measured the expression of 90 miRNAs in 79 breast cancer samples using RT-qPCR. We identified and quantified more than 3000 protein groups. We selected for subsequent analyses more than 1000 quantifiable proteins, defined as those identified at least in 75% of the samples in at least one type of sample with more than two unique peptides. Then, we analyzed the relations between miRNA and protein expressions. We identified miRNAs strongly related with processes considered as hallmarks of cancer, such as cellular adhesion, using probabilistic graphical models. Conclusions The integration of miRNA and protein expression patterns may be useful to describe how miRNAs regulate biological processes and signaling pathways in breast cancer. There is a need of new statistical approaches to evaluate these relations and to obtain meaningful information from such complex and massive data. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-07.

Optimization of an Immunohistochemical Method to Assess Distribution of Tight Junction Proteins in Canine Epidermis and Adnexae

Epidermal tight junctions (TJs) have been well characterized in human medicine. Abnormality of these structures is involved in skin diseases such as atopic dermatitis. There is little information about the expression and distribution of TJ proteins in the canine skin. The aim of this study was to develop an optimal immunohistochemical method for assessment of the expression of TJ proteins in the skin of healthy dogs. Formalin-fixed and paraffin wax-embedded skin biopsy samples from healthy human and canine patients were used. Canine skin samples were from the inguinal region and the nasal planum. Immunohistochemistry was used to study the expression of zonula occludens-1 (ZO-1), occludin and claudin-1, -4 and -7. Heat-induced antigen retrieval with EDTA (pH 9.0) yielded the best labelling of TJ proteins. ZO-1 and occludin were expressed in the cytoplasm and along the keratinocyte membrane, while claudin-1 and -4 were mainly membrane in distribution. ZO-1, occludin and claudin-1 were detected in all epidermal layers with the exception of the stratum corneum, while claudin-4 expression was restricted to the stratum granulosum. Expression of claudin-7 was difficult to evaluate. There was no difference in labelling pattern between inguinal and nasal planum skin.

Rapid Immunocytochemistry with Simple Heat-Induced Antigen Retrieval Technique for Improvement in the Quality of Cytological Diagnosis

Rapid immunocytochemistry (ICC) can improve the accuracy of intraoperative cytological diagnoses; however, it is usually applied without heat-induced antigen retrieval (HIAR). We established a HIAR method for rapid ICC and evaluated its efficacy and reliability. Rapidly fixed smear samples were immunostained using 35 antibodies. We compared the results of HIAR by boiling in a pot or heating in an electric kettle. The smears were incubated for 3 min with each primary antibody and immuno-enzyme polymer reagent, and for 1 min with diaminobenzidine solution. HIAR for 1 min using the kettle method yielded the best cellular integrity. For 32 out of the 35 antibodies, results achieved using rapid ICC within 11 min were comparable to that achieved using standard ICC. HIAR was essential for 13 antibodies. For two of the antibodies, HIAR was not required when standard ICC was applied, but consistent staining with rapid ICC was obtained only with HIAR. In conclusion, we established a rapid ICC procedure using a simple HIAR method, which allowed efficient immunostaining of a panel of antigens, including nuclear antigens, within only 11 min. The combined use of this rapid ICC technique with other staining techniques could be useful for improving intraoperative cytological diagnoses.

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.

Microproteomics by liquid extraction surface analysis: Application to FFPE tissue to study the fimbria region of tubo‐ovarian cancer

We have developed a new method for rapid analysis of a specific region on formalin fixed and paraffin embedded (FFPE) tissue sections. This method combines advantages of direct tissue MS analysis keeping histological information and conventional proteomics approaches for confident identification of proteins in complex sample. After histological annotation, heat-induced antigen retrieval is performed on FFPE tissue. Using a chemical inkjet printer, trypsin is deposited on discrete regions of less than 1 mm². After protein digestion, a liquid extraction is performed to retrieve all the peptides. Data coming from identification of proteins in cancer and benign region are compared. In total, 3649 unique peptides were identified (with a peptide strict false discovery rate less than 1%) corresponding to 983 and 792 nonredundant protein groups identified in benign and cancer region, respectively. A total of 123 protein groups are found only in cancer region and 315 are specific to the benign part. From these data, it has been possible to obtain different important signaling pathways involved in cancer processes and some proteins already known as biomarkers. This new approach using a combination of localized on-tissue protein digestion and liquid microextraction followed by LC-MS/MS analysis is useful for advancing our understanding of cancer biology. It is a rapid and innovative technique that will contribute positively to clinical proteomics.

Complete solubilization of formalin‐fixed, paraffin‐embedded tissue may improve proteomic studies

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.

Mismatch repair deficiency in colorectal cancer patients in a low-incidence area.

In a previous study we identified 206 patients with colorectal adenocarcinoma in the Northern Cape province of South Africa, diagnosed between January 2002 and February 2009. The age-standardised incidence was 4.2/100 000 per year world standard population. This is 10% of the rate reported in First-World countries. In high-incidence areas, the rate of abnormal mismatch repair gene expression in colorectal cancers is 2 - 7%. The aim of this study was to determine the prevalence of hMLH1- and hMSH2-deficient colorectal cancer in the Northern Cape. Formalin-fixed paraffin wax-embedded tissue blocks from 87 colorectal adenocarcinomas identified in the previous study were retrieved. Standard immunohistochemical staining methods were used to detect the expression of hMLH1 and hMSH2 (i.e. products of the hMLH1 and hMSH2 genes) in the tumours using heat-induced antigen retrieval and diaminobenzidene as a chromogen. Results. In 8 blocks there was insufficient tumour tissue and in 1 case the immunohistochemical staining failed, probably owing to poor fixation, leaving 78 cases for analysis. In 11 cases hMLH1 was deficient and in 6 cases hMSH2 was deficient. Overall, 21.8% of cancers were deficient for hMLH1 or hMSH2. Presuming that 80% of all hMLH1 deficiencies are due to hypermethylation of the gene, we found 10.5% of colorectal cancers in an area with a low incidence of colorectal cancer to be deficient in the product of the mismatch repair gene/s. This is approximately three times the reported rate in high-incidence areas.

Immunohistochemical expression of basement membrane laminin in histological grades of oral squamous cell carcinoma: A semiquantitative analysis

Background and Objectives:To determine the immunohistochemical (IHC) localization of basement membrane component laminin in histological grades of oral squamous cell carcinoma (OSCC). The purpose of this study was to demonstrate the loss of continuity of the basement membrane in OSCC using an antibody directed against laminin using advanced polymer staining system.Materials and Methods:A total of 30 cases of OSCC: 10 cases of well differentiated squamous cell carcinom (WDSCC), 10 cases of moderately differentiated squamous cell carcinoma (MDSCC), and 10 cases of poorly differentiated squamous cell carcinoma (PDSCC) were subjected to heat-induced antigen retrieval method using ethylene-di-amine-tetraacetic acid buffer in a microwave oven. Then the sections were stained with anti-laminin polyclonal antibody and visualized using super sensitive polymer horseradish peroxidase detection system. In each case, the integrity of the basement membrane laminin was assessed by using statistical analysis.Results:Statistical analysis showed a decreased distribution of laminin from WDSCC to MDSCC to PDSCC (P value 0.0573). The intracytoplasmic staining of laminin gradually increased from WDSCC to MDSCC to PDSCC (P value 0.0198).Interpretation and Conclusion:WDSCC cases showed more laminin expression in basement membrane around the tumor islands and less loss of continuity compared to MDSCC and PDSCC cases suggesting a greater enzymatic degradation of basement membrane components in MDSCC and PDSCC than WDSCC. The loss of structural basement membrane laminin and the presence of laminin in the tumor cells of PDSCC cases suggest that laminin helps in tumor invasion. The expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histologic differentiation and aggressiveness.

In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

BackgroundThe freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge.ResultsWe show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts.ConclusionThe modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.