Archival Clinical Samples Research Articles

Detection of viral induced double-stranded RNA intermediates in archival paraffin blocks

Double strand RNA (dsRNA) species were previously thought to be a ‘junk DNA’ equivalent are related to endogenous retroviral elements (ERV) inserted into the human genome. Genomic integration sites and blocking of these processes have been well documented in HIV and Hepatitis C related diseases. Viral integration into human genome has been shown in a range of solid tumors, but mechanisms less understood. The detection of dsRNA in the research setting has been largely limited to cell line models and controlled transfections. To better characterize function significance of ERV and interactions at the cellular level, we outline efforts to detect and quantify such viral genomic elements with a focus on archival clinical samples in the commonly stored paraffin block. Tissue blocks issues with clinical and biochemical documented viral infection and associated viral cytopathic changes were selected from the Department of Pathology and Biorepository and standard 4 micron sections were prepared. Two (2) commercially available antibodies raised to dsRNA fragments (J2 and K1 antibodies) were directly compared by chromogenic and immunofluorescent methods. In addition, downstream biomarkers as identified by literature searches include RIG-1 and IRF 7 as moderate probable success and MDA-5 and STAT 1 as higher level success. Correlative analysis of downstream pathway markers with labeling results of dsRNA antibodies suffers from lack of sensitive measures of dsRNA fragments as opposed to the downstream cellular pathway markers. This lack of concordance likely reflects the short nature of dsRNAs in decade old paraffin tissue and/or differences in abundance. Future work will focus on developing more sensitive probes or IF probe signal amplification. Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Double strand RNA (dsRNA) species were previously thought to be a ‘junk DNA’ equivalent are related to endogenous retroviral elements (ERV) inserted into the human genome. Genomic integration sites and blocking of these processes have been well documented in HIV and Hepatitis C related diseases. Viral integration into human genome has been shown in a range of solid tumors, but mechanisms less understood. The detection of dsRNA in the research setting has been largely limited to cell line models and controlled transfections. To better characterize function significance of ERV and interactions at the cellular level, we outline efforts to detect and quantify such viral genomic elements with a focus on archival clinical samples in the commonly stored paraffin block. Tissue blocks issues with clinical and biochemical documented viral infection and associated viral cytopathic changes were selected from the Department of Pathology and Biorepository and standard 4 micron sections were prepared. Two (2) commercially available antibodies raised to dsRNA fragments (J2 and K1 antibodies) were directly compared by chromogenic and immunofluorescent methods. In addition, downstream biomarkers as identified by literature searches include RIG-1 and IRF 7 as moderate probable success and MDA-5 and STAT 1 as higher level success. Correlative analysis of downstream pathway markers with labeling results of dsRNA antibodies suffers from lack of sensitive measures of dsRNA fragments as opposed to the downstream cellular pathway markers. This lack of concordance likely reflects the short nature of dsRNAs in decade old paraffin tissue and/or differences in abundance. Future work will focus on developing more sensitive probes or IF probe signal amplification. Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Molecular Classification of Bone Marrow Failure Syndromes: Protein Signatures As Surrogate Biomarker for Accurate Diagnosis of Severe Aplastic Anemia, Hypoplastic Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria Patients

Background/Purpose: Bone marrow failure syndrome is an example of disease entity where accurate diagnosis of Severe Aplastic Anemia (SAA), Paroxysmal Nocturnal Hemoglobinuria (PNH) and Hypoplastic Myelodysplastic Syndrome (MDS) is very challenging. The aim of this study was to identify panels of disease-specific /disease-associated proteins biomarkers to be used for more objective diagnosis and better prediction of disease prognosis of patients presenting with features of bone marrow failure syndromes.Methodology:Bone marrow plasma (MBP) and peripheral blood plasma (PBP) samples from 20 patients with bone marrow hypoplasia; including AA/MDS/PNH were subjected to expression proteome analysis using label-free quantitative liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).Results: Approximately 300 unique protein species were identified of which 107 and 218 were significantly differentially expressed (> 2- ∞- fold change &p < 0.05) in BMP and PBP respectively. These protein fingerprints independently discriminates patients into three distinct clusters; AA/MDS/PNH. Furthermore, only approx. 25% of the proteins were common between the two datasets from BMP and PBP. Some of the identified proteins were filtered and mapped using Ingenuity Pathway Analysis, and were associated with five different networks. The top two of these networks involved cell-to-cell signaling interaction, hematological system development and function, and immune cell trafficking. Only three of the differentially expressed proteins were uniquely expressed in SAA and MDS but absent in PNH, thus making these proteins potential biomarkers.The probable diagnostic utility of these proteins would be validated in large archival clinical samples. Our data indicates the utility of multivariate analysis of quantitative proteome data as a means of discovery of disease related or disease specific biomarkers for bone marrow syndromes.Conclusions:We have identified protein signatures capable of objective classification of bone marrow failure syndromes patients. Our expression proteomics strategy is very promising for identification of clinically useful biomarkers. These proteins once validated, on a larger cohort of patients, might be valuable to complement the currently existing parameters for reliable and objective disease diagnosis, monitoring treatment response and clinical outcome of bone marrow failure syndrome patients. DisclosuresOwaidah:King abdulaziz city for science, Novo Nordisk, Bayer: Honoraria, Research Funding.

Abstract 4748: Revealing the molecular portrait of triple negative breast tumors from an understudied population through omics analysis of formalin-fixed and paraffin-embedded tissues

Abstract Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal receptor 2 (HER2), is an aggressive form of breast cancer (BC) that is more prevalent in certain populations, in particular in low and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. We performed an integrative genomic analysis on FFPE samples from TNBC patients to identify molecular programs associated with this disease in an understudied population. We implemented whole exome sequencing of 12 tumor tissues and blood pairs, complemented with miRNA and mRNA profiling of the tumor tissues, using FFPE archived pathological samples from Mexican women. Sequencing analyses found TP53 and RB1 genes as the most frequently mutated. Transcriptional programs were characterized by the overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), the repression of cell cycle control pathways (TP53, RB1), the deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the utility of archival clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing the development of treatment strategies and of retrospective studies on the exploration of the geographic diversity of BC. Citation Format: Felipe Vaca-Paniagua, Rosa María Alvarez-Gomez, Carlos Perez-Plasencia, Hector Aquiles Maldonado-Martínez, Veronica Fragoso-Ontiveros, Federico Lasa-Gonsebatt, Luis Alonso Herrera, David Cantú, Enrique Bargallo-Rocha, Alejandro Mohar, Geoffroy Durand, Nathalie Forey, Catherine Voegele, Maxime Vallee, Florence Le Calvez-Kelm, James McKay, Maude Ardin, Stephanie Villar, Jiri Zavadil, Magali Olivier. Revealing the molecular portrait of triple negative breast tumors from an understudied population through omics analysis of formalin-fixed and paraffin-embedded tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2015-4748

High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.

In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%), APC (10.1%), PIK3CA (5.6%), KRAS (4.5%), SMO (3.4%), STK11 (3.4%), CDKN2A (3.4%) and SMAD4 (3.4%). Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.

Tissue proteomics and imaging mass spectrometry

Tissue proteomics and imaging mass spectrometry

Abstract 3213: A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays.

Abstract MUC16 and MSLN are tumor-specific antigens highly prevalent in ovarian and pancreatic cancers and have been used as targets in developing antibody-drug conjugates (ADCs). Development of a sensitive method to quantitatively measure these protein biomarkers in archival clinical samples may enable potential companion diagnostics to these novel therapeutic agents. Here we report the development of a method to quantitatively measure these biomarkers from formalin fixed paraffin embedded (FFPE) tumor samples using TaqMan proximity ligation assays (PLA). Monoclonal antibodies that recognize the extracellular domains of MUC16 and MSLN were conjugated to amplicon forming oligonucleotides to generate proximity ligation probes for TaqMan quantification. Using purified MUC16 and MSLN recombinant protein the assay sensitivities were determined to be lower than single-digit ng/mL concentrations for both MUC16 and MSLN and the dynamic ranges were &amp;gt;3 logs and ∼2 logs, respectively. In addition, the assays were demonstrated to be highly selective by testing lysates from expressing and non-expressing cell lines as determined by qRT-PCR. For clinical application of these assays, we further developed and optimized a simple two-step method for extraction and antigen retrieval of protein from archival FFPE tissues. FFPE extracts were prepared from a set of 10um sections of ovarian tumor tissue blocks and analyzed to determine relative MUC16 and MSLN protein expression levels. We benchmarked these results from the FFPE protocol with PLA data generated from matched fresh frozen tumor tissue as well as expression level determined by immunohistochemistry (IHC) and gene expression level determined by qRT-PCR assays from the same FFPE tissue blocks. All three comparisons with FFPE PLA were remarkably well correlated. In conclusion, we have established the technical feasibility and clinical utility of TaqMan PLA-based protein assays for sensitive and quantitative measurement of protein biomarkers in archival clinical samples. The methodology developed in this study represents a promising approach in companion diagnostic development for quantitative protein biomarker assessment. Citation Format: Lisa Ryner, Aimee Fourie, Siao Ping Tsai, Suzie Scales, Kathy Kozak, Ron Firestein, Yinghui Guan, An Do, Yulei Wang. A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2013-3213

Interleukin 28B genotype determination using DNA from different sources: A simple and reliable tool for the epidemiological and clinical characterization of hepatitis C

Recent studies reported a close correlation between polymorphisms in the Interleukin (IL)28B gene and rates of resolution of hepatitis C virus infection occurring spontaneously or induced by treatment. The diagnostic utility of IL28B genotype, however, is not understood completely. For rapid data collection on the natural history of HCV infection in patients with different IL28B genotype, simple, sensitive and rapid methods suitable for non-invasive and archival clinical samples are needed urgently.A real-time polymerase chain reaction (PCR) method for IL28B typing (rs12979860) was developed using very small DNA quantities extracted from different biological specimens.Consistent IL28B genotyping of at least two DNA samples obtained from different sources such as whole blood, buccal swab, serum, and formalin fixed paraffin-embedded liver tissue was obtained from 58 patients with liver disease of mixed etiology. IL28B genotype prevalence in 170 patients with liver disease in this region of Italy was consistent with data reported in Caucasian populations. Differential distribution of genotypes was observed according to response to treatment in 68 patients infected with HCV, with higher prevalence of CC genotype in responders (50%) compared to non-responders (17.85%; p=0.015). These results indicate that the possibility of reliable IL28B genotyping using different DNA sources may represent a useful tool for both clinical research and characterization of patients with hepatitis C.

Protein phosphorylation analysis in archival clinical cancer samples by shotgun and targeted proteomics approaches

Protein phosphorylation affects most eukaryotic cellular processes and its deregulation is considered a hallmark of cancer and other diseases. Phosphoproteomics may enable monitoring of altered signaling pathways as a means of stratifying tumors and facilitating the discovery of new drugs. Unfortunately, the development of molecular tests for clinical use is constrained by the limited availability of fresh frozen, clinically annotated samples. Here we report phosphopeptide analysis in human archival formalin-fixed, paraffin-embedded (FFPE) cancer samples based on immobilized metal affinity chromatography followed by liquid chromatography coupled with tandem mass spectrometry and selected reaction monitoring techniques. Our results indicate the equivalence of detectable phosphorylation rates in archival FFPE and fresh frozen tissues. Moreover, we demonstrate the applicability of targeted assays for phosphopeptide analysis in clinical archival FFPE samples, using an experimental workflow suitable for processing and analyzing large sample series. This work paves the way for the application of shotgun and targeted phosphoproteomics approaches in clinically relevant studies using archival clinical samples.

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections.

Tissue microarray – a high‐throughput molecular analysis in head and neck cancer

With the identification of a number of novel markers having diagnostic, prognostic, and therapeutic significance, the application of tissue microarray (TMA) has become a valuable tool for validating candidate markers in cancer research. The TMA is a high-throughput technique, which allows large-scale analyses of hundreds of archival clinical tissue samples using the 'array' approach. This paper highlights briefly its robust technology, technical aspects of its construction, and the validity of the TMA results for oral pathology diagnostics by reviewing data from recent literature particularly with reference to head and neck cancer.

Fluorescence Single-Molecule Counting Assays for High-Sensitivity Detection of Cytokines and Chemokines

A recent focus in clinical immunodiagnostics is to improve sensitivity of assays for rare circulating protein biomarkers. The ability to detect low concentrations of certain biomarkers translates to diagnosis of disease at an earlier stage, which can positively impact prognosis and disease management (1)(2)(3). In research (for example in the discovery and development of new biomarkers), targeted assays for individual proteins are often required before multiplexing strategies are developed. Cross-reactivity of antibodies often compromises assay sensitivity in multiplexed immunoassays(4). Ultrasensitive individual assays consume only small sample volumes, a characteristic that can be advantageous in the case of scarce archival clinical samples. The development of single-molecule detection approaches has provided new opportunities for improving immunoassay sensitivity and miniaturization (5). Fluorescence confocal microscopy with laser-induced excitation enables single-molecule detection in extremely small interrogation zones (on the order of femtoliters). By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background(6). Counting these bursts, or molecular events, by applying fluorescence thresholds on the basis of the internally derived background noise generates experimental data whose precision is dictated by counting statistics. Hence, sample read times can be adjusted to achieve a desired counting precision. The combination of small interrogation volumes and short sample read times can also lead to rapid analysis. Despite the advantages of single-molecule detection and the development of single-molecule detection instruments, adaptation of this technology to quantification of serum or plasma analytes has only scarcely been reported in the literature [for example, (7)]. Building on our previous work(8), we constructed single-molecule immunoassays (SMIAs) that use immune capture of analytes on microparticles, tagging with biotinylated antibodies, and quantification of fluorophore-labeled …

Molecular analyses and prognostic relevance of HPV in head and neck tumours

HPV involvement in head and neck (HN) cancer is still under active investigation. Fresh frozen and archival clinical samples from 115 patients affected by HN carcinomas were analysed by PCR-based methods and direct sequencing. HPV types, intra-type variants, physical status, viral load and viral transcript presence were determined. HPV positivity was correlated with the main clinical-pathological features, including smoker and drinker status, and the clinical outcome. Twenty-one tumours were HPV positive (18.3%) with HPV16 being the most frequent type (n=14) followed by HPV6 (n=4), HPV33, HPV35, and HPV58 (n=1, each type). Tonsil carcinomas contained more high-risk HPV types (6/8; 75%) than all other sites (p=0.0004). HPV16 genome was integrated in all analysed tumours, as pure integrated form or mixed with concomitant episomal forms (4 cases). The viral load showed a wide variability (range, 0.7-485 copies per cell) with the highest value detected in a larynx tumour and the lowest one in a case of cancer of the oral cavity. In 9 HPV-positive samples where mRNA was available, transcripts of viral early oncogenes originating by integrated, episomal or mixed forms of the viral genome were found. A statistically significant correlation was evidenced between HPV and tumour differentiation, being the virus more associated with tumour grade G3/G4. Multivariate Cox regression analysis revealed that lymph-node and grade status were significant independent factors for a worse disease-free survival and overall survival, whereas the HPV status was associated with a better overall survival (OR, 0.33; 95% CI, 0.13-0.81; p=0.01). Taken together these results indicate that distinct pathological mechanisms for the malignant transformation in each single HN subsite should be taken in account; HPV molecular analyses should be considered a valid tool to distinguish subsets of oropharyngeal tumours and HPV presence could be useful for the prognostic assessment of HNSCC.

Tissue Microarray Applications: A Valuable Clinical and Research Resource

Widespread applications of tissue microarrays (TMAs) in both cancer research and clinical pathology demonstrate a versatile and portable technology. The rapid integration of TMA technology into the biomarker discovery and validation process reflects the forward thinking of researchers who pioneered the high-density TMA. The precise arrangement of hundreds of archival clinical tissue samples into a composite paraffin block is now a proven method for efficient and standardized analyses of molecular markers. With applications in cancer research, TMAs are a valuable tool in viiidating candidate markers identified in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, TMAs are used widely in immunohistochemistry for quality control and quality assurance. TMAs can demonstrate antibody thresholds on a single slide, which aid in optimization where high and low signal intensities can be seen. This type of application lends itself to clinical pathology and immunohistochemistry panels run daily to improve biomarker identification and characterization. The TMA industry now includes public and private resources with varying degrees of research utility. In this article, we will explore several potential TMA applications in basic research, prognostic oncology, and drug discovery. (The J Histotechnol 28:223, 2005)Submitted September 6, 2005; accepted with revisions December 6, 2005

Tissue Microarray Applications: A Valuable Clinical and Research Resource

Widespread applications of tissue microarrays (TMAs) in both cancer research and clinical pathology demonstrate a versatile and portable technology. The rapid integration of TMA technology into the biomarker discovery and validation process reflects the forward thinking of researchers who pioneered the high-density TMA. The precise arrangement of hundreds of archival clinical tissue samples into a composite paraffin block is now a proven method for efficient and standardized analyses of molecular markers. With applications in cancer research, TMAs are a valuable tool in viiidating candidate markers identified in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, TMAs are used widely in immunohistochemistry for quality control and quality assurance. TMAs can demonstrate antibody thresholds on a single slide, which aid in optimization where high and low signal intensities can be seen. This type of application lends itself to clinical pathology and immunohistochemistry panels run daily to improve biomarker identification and characterization. The TMA industry now includes public and private resources with varying degrees of research utility. In this article, we will explore several potential TMA applications in basic research, prognostic oncology, and drug discovery. (The J Histotechnol 28:223, 2005)Submitted September 6, 2005; accepted with revisions December 6, 2005

Human Leukocyte Class I Antigen and ??2-Microglobulin Expression in Conjunctival Dysplasia, Carcinoma In Situ, and Squamous Cell Carcinoma

Malignant transformation of cells is frequently associated with abnormalities in HLA expression. These abnormalities may play a significant role in the clinical course of the disease because the cellular immune response to tumors relies on concomitant recognition of tumor antigens with self-HLA molecules. Since HLA molecules mediate interactions of tumor cells with specific receptors on T and natural killer (NK) cells, tight control of expression of HLA molecules is critical for initiation and implementation of an effective cellular immune response. There is not much information on HLA class I antigens in conjunctival carcinomas. We studied the immunoexpression of HLA class I antigen and beta2-microglobulin (beta2-m) in conjunctival dysplasias and conjunctival squamous cell carcinomas. HLA class I antigen and beta2-m expression were analyzed in 10 conjunctival dysplasias, 6 carcinomas in situ, and in 11 conjunctival carcinomas by immunoperoxidase staining with monoclonal antibodies to HLA class I antigen and beta2-m on the archival clinical samples. Immunoanalysis was done according to the International Histocompatibility Working Group Project Description. HLA class I antigen and beta2-m were heterogeneous in 10 conjunctival dysplasias, negative in 6 carcinomas in situ, and 11 conjunctival squamous cell carcinomas. The difference of immunoexpression for HLA class I and beta2-m among the 3 groups was statistically significant (P <0.001). HLA class I antigens and beta2-m are decreased in conjunctival dysplasias and negative in carcinomas in situ, and carcinomas. The findings support the role of cytotoxic T lymphocyte-mediated control of tumor growth in the clinical course of conjunctival squamous cell carcinomas.

Tissue microarrays: applications in genomic research

The widespread application of tissue microarrays in cancer research and the clinical pathology laboratory demonstrates a versatile and portable technology. The rapid integration of tissue microarrays into biomarker discovery and validation processes reflects the forward thinking of researchers who have pioneered the high-density tissue microarray. The precise arrangement of hundreds of archival clinical tissue samples into a composite tissue microarray block is now a proven method for the efficient and standardized analysis of molecular markers. With applications in cancer research, tissue microarrays are a valuable tool in validating candidate markers discovered in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, tissue microarrays are used widely in immunohistochemistry quality control and quality assurance. The timeline of a biomarker implicated in prostate neoplasia, which was identified by complementary DNA expression profiling, validated by tissue microarrays and is now used as a prognostic immunohistochemistry marker, is reviewed. The tissue microarray format provides opportunities for digital imaging acquisition, image processing and database integration. Advances in digital imaging help to alleviate previous bottlenecks in the research pipeline, permit computer image scoring and convey telepathology opportunities for remote image analysis. The tissue microarray industry now includes public and private sectors with varying degrees of research utility and offers a range of potential tissue microarray applications in basic research, prognostic oncology and drug discovery.

HLA class II antigen expression in conjunctival precancerous lesions and squamous cell carcinomas

Purpose. The expression of human leukocyte antigen (HLA) class II molecules on the cell surface is necessary for the presentation of peptide antigens to helper CD4 T lymphocytes of the immune system. We studied the immunoexpression of HLA class II antigen in conjunctival precursor lesions and conjunctival squamous cell carcinomas. Methods. HLA class II antigen expression was analyzed in 8 conjunctival dysplasias, 6 carcinomas in situ and in 7 conjunctival squamous cell carcinomas, by immunoperoxidase staining with monoclonal antibody to HLA class II antigen on the archival clinical samples. Immunoanalysis was done by a semi quantitative method based on the intensity of staining and the percentage of stained cells. Results. HLA class II antigen immunoexpression was heterogeneous in 8 conjunctival dysplasias and in 6 carcinoma in situ and negative in 7 conjunctival squamous cell carcinomas. Conclusions. Human leukocyte class II antigen immunoexpression is decreased in conjunctival precancerous and squamous cell carcinomas.

Standardization of a single-cell assay for sensitive detection of multidrug resistance protein expression in normal and malignant cells in archival clinical samples

Multidrug resistance protein (MRP), like P170, confers multidrug resistance, but its clinical relevance is uncertain, whereas P170 is an accepted cause of chemotherapy failure for which ongoing reversal trials are being conducted. Because such trials have been only modestly successful, we must investigate alternative drug resistance mechanisms such as MRP, which is poorly blocked by P170 inhibitors. The significance of MRP has remained undefined because MRP mRNA is difficult to assay in archival material, does not necessarily reflect MRP levels, and is widely expressed in normal or hematopoietic cells within tumors and bone marrow. Because conventional immunoblot or immunocytochemistry may not be sensitive enough to detect low or heterogeneous MRP expression in clinical samples, we elected to score MRP in single tumor cells by modifying our P170 assays that have proven valuable for correlating P170 expression with the outcome of pediatric cancer chemotherapy. We enhanced the signal-to-noise ratio with several peroxidasetagged secondary antibody layers and staining refinements, standardizing the assay with MRP-negative and MRP-positive but P170-negative transfected or drug-selected controls in which MRP was quantified by immunoblot. We confirmed sensitivity by staining a very low MRP-expressing revertant line and “mixed” samples containing small numbers of positive cells; we confirmed specificity by applying two antibodies directed against separate MRP epitopes. We examined neuroblastoma, osteosarcoma, rhabdomyosarcoma, and retinoblastoma samples, identifying MRP-positive malignant cells, which were distinguishable from MRPpositive normal cells. This assay may be valuable for early diagnosis of low but potentially important MRP expression, which would allow timely application of alternative therapy, perhaps with MRP-specific blockers.