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Mrna In-Situ Hybridization Using Biotinylated Oligonucleotide Probes: Implications for the Diagnostic Laboratory* JULIANA G

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Mrna In-Situ Hybridization Using Biotinylated Oligonucleotide Probes: Implications for the Diagnostic Laboratory* JULIANA G ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 24, No. 4 Copyright © 1994, Institute for Clinical Science, Inc. mRNA In-situ Hybridization Using Biotinylated Oligonucleotide Probes: Implications for the Diagnostic Laboratory* JULIANA G. SZAKACS, M.D.t and SANDRA K. LIVINGSTON, HTL (ASCP)tt tDepartment of Pathology, tMolecular Histology Laboratory University of South Florida, College of Medicine Tampa, FL 33612 ABSTRACT It is now possible to detect low copy numbers of messenger ribonucleic acid (mRNA) while retaining good histologic morphology for the determi­ nation of specific gene expression in diseased tissues. This technology will allow the pathologist to provide important prognostic information about tumors (expression of oncogenes and growth factors), to identify the sub­ clones within the tumor which may be most likely to metastasize (expres­ sion of adhesion molecules and proteases) and to identify etiologic genetic aberrations (viral insertions). A technique for in-situ hybridization to mRNA has been developed for use in formalin fixed paraffin embedded tissues which is suitable for a hospital histology laboratory. Optimal con­ ditions for the procedure were determined by using a biotinylated poly (d)T oligonucleotide probe. Results were dependent on the tissue type, fixation time, condition of the tissue prior to fixation, and degree of diges­ tion before hybridization. The temperature and conditions of hybridization were optimized so that the poly d(T) control probe and the longer test probe could be run simultaneously. Streptavidin and avidin alkaline phos­ phatase detection systems were tested using levamisole to minimize back­ ground staining, and a biotin blocking agent to reduce reaction to renal tubular biotin. Increasing the temperature of stringency washes did not significantly improve the specificity but had a markedly detrimental effect on tissue morphology. The mRNA appears to remain stable within routinely fixed surgical mate­ rial over long periods of time allowing for large retrospective studies. A review of c-erbB-2 expression in 16 human breast lesions was carried out comparing mRNA in-situ hybridization to immunoperoxidase and cytosolic methods. By direct localization of both message and antigen, it was possi­ ble to demonstrate focal positivity that cytosolic methods did not detect. Aberrant translation was noted in one case, and c-erbB-2 expression in non-malignant breast was detected in two cases. * Send reprint requests to Juliana G. Szakacs, M.D., Department of Pathology, White River Junction VAM & ROC, White River Junction, VT 05009. 324 0091-7370/94/0700-0324 $02.00 © Institute for Clinical Science, Inc. mRNA IN-SITU HYBRIDIZATION 325 Introduction more sensitive in certain cases than Northern blotting when only a small number of cells are expressing the mes­ Routine molecular histology is now sage in a total quantity too low to detect commercially available in kit form for on a blot. deoxyribonucleic acid (DNA) in-situ Gene activation results in the forma­ hybridization (ISH) to viral genes, and tion of heterologous nuclear RNA recently kits and nonradioactive detec­ (hnRNA) by RNA polymerase II. Within tion systems geared to mRNA in-situ the nucleus a 3'-poly A tail (composed of hybridization have been developed up to 300 adenylyl residues) is added to which greatly simplify this process. Bio- the hnRNA along with a 5'-methyl cap. tinylated oligonucleotide probes have The RNA then undergoes splicing which been found to be both highly sensitive removes the intron or non-coding and specific,1 eliminating the need for sequences resulting in mature mRNA special radiation licenses, waste disposal, which is transported from the nucleus to and the need to label probes fresh with the cytoplasm for translation. The aver­ each run. Biotinylation of probes can be age cell contains approximately 360,000 performed enzymatically in the labora­ molecules of mRNA which are divided tory or oligonucleotide probes can be into 10,000 to 20,000 different species. synthesized using biotinylated nucleo­ This means that many specific messages tides. These remain stable for 6 to 12 will be found in low numbers, less than months. The use of shorter oligonucleo­ 30 molecules per cell.3 Since the majority tide probes as opposed to longer probes of mRNA contains a poly A tail, a poly raised in plasmids has not compromised d(T) probe was used to detect mRNA specificity, and they are much easier to preservation in fixed tissues and to opti­ obtain. They are single stranded DNA mize fixation, digestion, and hybridiza­ which means that no dénaturation step is tion parameters for varying tissue types.3 required prior to hybridization, and there Once this is accomplished, studies of is no decrease in efficiency owing to specific messages may be undertaken. sense strands binding to probe. The These may include studies for the pres­ selection of the probe sequence, how­ ence of oncogene expression for correla­ ever, becomes important in shorter tion with protein expression studies probes to ensure specificity. Computer (c-erbB-2 in breast carcinoma is our programs are now available to screen example) or the development of probes selected sequences against all known for the study of protein products for genetic sequences. These programs will which there is no available antibody or identify the probability of hybridizing to for tissues in which the protein epitopes the selected message and to any other are lost during processing. In-situ genetic material with similar sequences. hybridization to mRNA will allow detec­ It has now been shown by correlation tion of gene activation even if protein is of in-situ hybridization and northern blot not expressed owing to malfunctions of analysis that ISH, using non-radioactive translation; however, correlation with the labels, is sensitive enough to detect as protein product will provide definitive few as 10 molecules of mRNA per cell knowledge of gene activation and protein with some authors claiming the ability to synthesis within a cell type. detect one copy per cell.2 In addition, The identification of oncogene ampli­ ISH will define the specific cell type or fication and expression in tumors is subclone which is the source of the mes­ becoming the standard of care. The sage within the tissue sample and may be immense field of molecular diagnostics is 326 SZAKACS AND LIVINGSTON making its way into the diagnostic labo­ anti-c-erbB-2 immunohistochemistry ratory and soon therapies will be based and the cytosolic measurement of on the presence or absence of onco­ the oncoprotein. genes, drug resistance genes or muta­ tions which have been found to cor­ relate with the progression of disease, Materials and Methods response to therapy and survival. Excel­ lent reviews have been prepared by RNAse free deionized water was pre­ Richardson4 and Yamamoto.5 pared by the addition of 1 ml/L diethyl Within the last few years, oncologists pyrocarbonate (DEPC, Sigma D5758)*, have been looking for prognostic indica­ with overnight incubation followed by tors which will help them determine the autoclaving to remove DEPC. The need for adjuvant therapy in node nega­ DEPC water was used for all steps prior tive breast cancer patients with small pri­ to and including hybridization. All glass­ mary tumors. Although 80 to 90 percent ware was baked at 200°C for a minimum of these patients do well with lumpec­ of four hours to destroy RNAses, and ster­ tomy and radiation alone, a small number ile pipette tips and plasticware were would derive significant benefit from employed where possible. adjuvant chemotherapy. In an attempt to identify this group of patients, a number of tumor parameters are being studied T is s u e s and have been associated with poor prog­ nosis. These include: aneuploidy, high Fischert 344 rat tissues were used to cycling index, lack of estrogen and pro­ determine optimum fixation and diges­ gesterone receptors, and increased epi­ tion times for the poly d(T) probe and to dermal growth factor receptor (EGFr), establish the procedure. Under general cathepsin-D, p53, and c-erbB-2.6,7 How­ anesthesia, rats were exsanguinated and ever, no single finding is predictive of all tissues were sliced into 5 mm sections outcome on a case by case basis. Most and immediately placed in 10 percent studies of c-erbB-2 (also known as HER2/ buffered formalin for 4 to 72 hours. neu) have been done by protein analysis Screened tissues included: brain, spinal in cytosolic extractions from tumor sam­ cord, eye, tongue, trachea, esophagus, ples8,9,10 or quantification of DNA ampli­ lungs, heart, aorta, liver, muscle, skin, fication by Southern blotting.11,12,13 In stomach, small and large bowel, kidney, both of these techniques, normal and adrenal, bladder, prostate, and testes. malignant cell populations within the Sixteen paraffin blocks of human breast sample are studied, which could result in tissue removed surgically for diagnosis at a small subclone of highly malignant the Moffitt Cancer Center! were tested cells expressing the oncogene being for c-erbB-2 mRNA and oncoprotein. diluted out. Very few correlations of gene These tissues were handled routinely, amplification, expression, and translation and fixation varied from 12 to 72 hours within one tumor have been carried out. prior to processing. Early results suggest that these parame­ ters may not always be concordant.14 Which, if any, of these parameters is most * Sigma Chemical Company, P.O. Box 14508, St. closely associated with tumor progres­ Louis, MO 63178. t Fischer 344 rats, % Charles River Breeders, sion and survival is not known. A study Kingston, NY 12401. has been initiated by us to correlate t Moffitt Cancer and Research Center, 12902 in-situ hybridization to mRNA with Magnolia Dr. Tampa, FL 33612. mRNA IN-S1TU HYBRIDIZATION 327 Q uantitation o f So l u t io n s c-e r b B-2 O n c o p r o t e in 20X SSC ph 7.0 3M NaCl, 0.3 M trisodium citrate Fresh frozen tissue samples were sent 50X Denhardt’s Solution to Dianon Systems § for cytosolic quanti­ Ficoll 400 5 gm fication of c-erbB-2 oncoprotein at the Polyvinyl pyrrolidine 5 gm BSA 5 gm time of surgery.
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