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In situ amplification: its application to diagnostic pathology
Current Diagnostic Pathology (1996) 3, 201-210 9 1996 Pearson ProfessionalLtd
Mini-symposium: Advances in diagnostic technology
In situ amplification: its application to
diagnostic pathology
J. J. O'Leary, M. M. Kennedy, R. J. Landers and J. O'D. McGee
initially described technique of in situ PCR, to include PRINS (primed in situ labelling), cycling PRINS, in situ PNA PCR (peptide nucleic acid PCR), in situ PNA PCR, IS-Taq Man PCR and allele specific amplification.
INTRODUCTION Solution phase polymerase chain reaction (PCR) has become one of the most revolutionary tools in molecular pathology. The technique, conventionally performed in a tube, permits the amplification of single copy mammalian/viral targets (either DNA or RNA) in fresh/frozen and archival paraffin-embedded material. However, the inability to visualize and localize amplified product within cells and tissue specimens has been a major limitation, especially for pathologists attempting to correlate genetic events with pathological changes. In situ hybridization (ISH) has partly addressed this problem by permitting localization of specific nucleic acid sequences at the individual cell level. However, most conventional non-isotopic in situ detection systems do not detect single copy genes, except for those incorporating elaborate sandwich detection techniques as described in 1992 by Herrington.l Before amplification can be performed in solution phase PCR (SPPCR), nucleic acid extraction is firstly carried out, which necessitates cellular destruction. Subsequent correlation of results with histological features is consequently not possible. Recently, a number of studies have described 'hybrid' techniques coupling PCR with in situ hybridization. 2-~4 The techniques have not been universally accepted because of technological problems encountered during amplification of the desired nucleic acid sequence. However, in recent months, dedicated equipment and chemistry have greatly facilitated the performance of in situ amplification and are rapidly expanding the potential use of these tools in diagnostic pathology. The repertoire of in situ amplification has now been extended to include a number of modifications of the
GENERAL
PRINCIPLES
All of the above techniques attempt to create doublestranded or single-stranded DNA amplicons within the cell, which can either be detected directly or following an in situ hybridization step. For successful application of the techniques, the aim is to achieve a fine balance between adequate digestion of cells (allowing access of amplification reagents) and maintaining localization of amplified product within the cellular compartment while preserving tissue/cell morphology. To do this, the techniques require specific cyclical thermal changes to occur at the individual cell level, akin to that which occurs in solution phase PCR. These changes initially bring about denaturation of double-stranded DNA (dsDNA) to single-stranded form (ssDNA), if a DNA target is being amplified. For RNA target specific amplification, the RNA template is already single stranded and reverse transcription is carried out to create a cDNA template. Specific short runs of oligonucleotides (primers) are then annealed to the respective ends of the desired target sequence and a thermostable enzyme (Taq DNA polymerase or Klenow fragment) is used to extend or ligate (DNA ligase, in IS-LCR; see below) the correctly positioned primers. Subsequent rounds of thermocycling then increase the copy number of the desired target sequence, in a nucleic acid amplification reaction. However, unlike solution phase PCR, an exponential increase in the amount of amplified product is never achieved. Instead, linear amplification occurs in most situations because of the relative inefficiency of the techniques; because of the compactness of the nuclear compartment of the cell
J. J. O'Leary, M. M. Kennedy, R. J. Landers and J. O'D. McGee,
Nuffield Departmentof Pathologyand Bacteriology,Universityof Oxford, OxfordOX3 9DU, UK 201
202 CURRENTDIAGNOSTICPATHOLOGY (containing dsDNA ssDNA pre-mRNA and histone proteins), there are problems of accessibility of amplification reagents to the desired nucleic acid sequence. Once the amplicon is created, detection must then be carried out. If the investigator has utilized a labelled primer or nucleotide, then the amplicon is labelled and can be demonstrated directly by immunocytochemical techniques. Alternatively, an in situ hybridization step (with a single-stranded oligoprobe or a double-stranded genomic probe) is carried out post-amplification, adhering to the general rules of standard in situ hybridization. Definitions Several essentially similar techniques are now described as follows.
DNA in situ PCR (IS-PCR) PCR amplification of cellular DNA sequences in tissue specimens using a labelled nucleotide (i.e. dUTP) within the PCR reaction mix. The labelled product is then detected by standard detection techniques as for conventional in situ hybridization or immunocytochemistry (Fig. 1).
Labelled primer driven in situ amplification (LPDISA) In situ amplification of DNA sequences using a labelled primer within the PCR reaction mix. The labelled product is then detected as for DNA IS-PCR. PCR in situ hybridization (PCR-ISH) PCR amplification of cellular DNA sequences in tissue specimens followed by in situ hybridization detection of
the amplified product with a labelled internal or genomic probe. The labels used can either be isotopic (p3z, $35) or non-isotopic (e.g. biotin, digoxigenin, fluorescein) (Fig. 1).
Reverse transcriptase in situ PCR (RT in situ PCR) The amplification of mRNA sequences in cells and tissue specimens by firstly creating a copy DNA template (cDNA) with RT and then amplifying the newly created DNA template as for DNA IS-PCR.
Reverse transcriptase PCR in situ hybridization (RT PCR-ISH) Amplification of RNA sequences in cell and tissue specimens by creating a cDNA template with RT. The newly created cDNA is then amplified, and the amplicon probed with an internal oligonucleotide as in PCR-ISH.
PRINS (primed in situ amplification) and cycling PRINS Amplification of specific genetic sequences in metaphase chromosome spreads or interphase nuclei, using one primer to generate single-stranded PCR product. If many rounds of amplification are utilized then the technique is called cycling PRINS (Fig. 2A and B). 33 In situ peptide nucleic acid PCR (IS-PNA-PCR) and PCR-PNA-ISH
IS-PNA-PCR. The amplification of DNA targets using a DNA mimic molecule, PNA. PNA is a simple molecule, made up of repeating N-(2-aminoethyl)-glycine units linked by amide bonds. Purine (A and G) and pyrimidine
Fig, 1--Schematic representation of in situ PCR and PCR-ISH.
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Fig. 2--Schematic representation of (A) PRINS and (B) cycling PRINS by a single primer approach.
bases (C and T) are attached to the back bone by methylene carbonyl linkages. PNA is not elongated by Taq DNA polymerase when used as a primer and thereforc can be used in primer exclusion assays, which allows the discrimination of point mutations and direct individual cell haplotyping. PCR-PNA-ISH. In this second reaction which employs PNA, a 15-20 mer PNA probe is used for the in situ
hybridization step following amplification. PNAs have higher melting temperatures (Tms) than DNA oligoprobes and single point mutations in a PNA-DNA duplex lower the Tm by approximately 15~ as compared to the corresponding DNA-DNA mismatch duplex. 45 IS-TaqMan PCR
Amplification of DNA sequences using a conventional primer pair as in standard PCR. However, an internal TaqMan probe is added to the amplification mix. A flourescent reporter molecule (FAM, HEX, etc.) is placed at the 5' end of the probe. At the 3' end, a quencher molecule (again fluorescent, usually TAMRA) is positioned. Once the probe is linearized and intact, the proximity of the quencher to the reporter molecule does not allow any fluorescence from the reporter molecule.
Tat} DNA polymerase possesses two properties important for the reaction: (1) Y-strand fork displacement (which allows Taq DNA polymerase to lift off a single strand in Y configuration and (2) 5'-3" endonucleolytic activity, which causes cleavage of the linker arm which attaches the reporter molecule to the 5' end of the Taq Man probe (Fig. 3), thereby giving rise to fluorescence if specific amplification has o c c u r r e d . 46'47 In situ allele specff)'c amplification In situ allele specific amplification (IS-ASA) technique
utilizes ARMS PCR (amplification refi'actory mutation system) which has the ability to detect point polymorphisms in human DNA sequences using artificially created base pair mismatches at the 3' end of PCR primers. If the polymorphism matching that of the primer sequence is present, amplification of that sequence wilt preferentially occur.
AMPLIFICATION OF DNA TARGETS IN CELLS AND TISSUES Equipment Several different types of equipment can be used for in situ amplification, ranging from standard thermal cycters
204 CURRENTDIAGNOSTICPATHOLOGY
Fig. 3--Schematic representation of Taq Man PCR (see text for details of method).
(with modifications), thermocycling ovens and specifically dedicated thermocyclers (Perkin Elmer Gene Amp in situ PCR system 1000, Hybrid Omnigene/Omnislide and MJB slide thermocycler). If a standard thermal cycler is used, then an amplification chamber initially must be created for the slide. This is usually made from aluminium (aluminium foil boat). This boat containing the slide is placed on the thermal cycler, covered with mineral oil and then wrapped completely. However, optimization of thermal conduction is never completely achieved. 'Thermal lag' (i.e. differences in temperature between the block face, the glass slide and the PCR reaction mix at each temperature step of~ the reaction cycle) is commonly encountered. 19 This has been addressed by the newer in situ amplification machines, which offer in-built slide temperature calibration curves with greater thermodynamic control.
Starting material In 1990, Haase initially described in situ PCR in intact fixed single cells, suspended in PCR reaction buffer. After amplification, cells were cytocentrifuged on to glass slides and the amplified product was detected by in situ hybridization. 2 Other early investigators used pieces of glass slides (with cells from cytocentrifuge preparations) in standard ependorf tubes, incubated directly in PCR reaction buffer. 1~ More recently techniques using tissues and cells attached to microscope slides have been described with amplification carried out either on heating blocks or in cycling ovens. 3~4 For IS-PCR, PCR-ISH, IS-LCR, IS-PNA PCR and IS-TaqMAN PCR, fixed cells and tissues - including archival paraffin-embedded material - can be used for
amplification. Best results are obtained with freshly fixed cells and tissues, although successful amplification with old archival material (up to 40 years) has been achieved (O. Bagasra, personal communication). Fixed metaphase chromosome spreads and interphase nuclei can be used for the detection of specific subchromosomal regions using PRINS and cycling PRINS. 33 For successful in situ amplification to occur, a rigid cellular cytoskeleton must be created which provides a suitable micro-environment allowing access of amplification reagents with minimal leakage of amplified product. Satisfactory results may be obtained with tissues fixed in 1-4% paraformaldehyde, neutral buffered formaldehyde (NBF) and 10% formalin (12-24h for biopsy/solid tissue; 10-30 min for cytological preparations). 16J8 Less consistent results are obtained with ethanol and acetic acid fixed t i s s u e s . 16 Fixation of cells with formaldehyde fixatives produces a number of drawbacks. Formaldehyde is not easily removed from tissues, even after tissue processing. Aldehyde groups react with DNA and histone proteins to form DNA-DNA and DNA-histone protein cross-links. 48 Formaldehyde fixation also 'nicks' DNA template (random breaks in dsDNA), which may be non-blunt ended (i.e. non-overlapping ends). These nicks may subsequently act as potential priming sites for Taq DNA polymerase leading to incorporation and elongation of labelled and unlabelled nucleotides (i.e. dATP, etc.) analogous to in situ end labelling for apoptosis. This process occurs at room temperature, leading to spurious results with DNA IS-PCR. Because repeated cycles of heating and cooling are used during in situ amplification, it is imperative that cells and tissues are adequately attached to a solid support (usually glass), so that detachment does not
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occur. Glass slides are pre-treated with coating agents to ensure maximal section adhesion, the most commonly used agents being aminopropyltriethoxysilane (APES), Denhardt's solution and Elmer's glue.
Cell and tissue permeabilization Cells must be adequately digested and permeabilized to facilitate access of reagents. This is usually achieved by protease treatment (e.g. proteinase K, pepsin or trypsin) and/or mild acid hydrolysis (0.01 N-0.1 N HC1). While extended proteolytic digestion is performed for solution phase PCR (often up to 24--48 h) to overcome DNAhistone protein cross-linking, digestion of cells and tissues for in situ amplification is limited by the necessity of maintaining cell morphology, architecture and cell/ tissue adhesion. Maximal digestion times and protease concentrations have to be optimized for the particular tissue/cytological preparation employed. Usual working conditions at 37~ are as follows: 0.1 mg/ml proteinase K for 15-25 rain for fixed tissues; 0.01-0.1 mg/ml proteinase K for up to 5 rain for cytological preparations. Longer digestion times inevitably compromise cellular morphology, but as a result incomplete dissociation of histone protein-DNA cross-links occurs, hindering the progression of Taq DNA polymerase along the native DNA template. Acid hydrolysis probably acts by driving such cross-links to complete dissociation. Alternatively, microwave irradiation of cells and tissue sections can be used to expose nucleic acid templates. Short pulses of microwave irradiation (with or without proteolysis) with a citrate buffer analogous to antigen retrieval for immunocytochemistry - effectively allow access of amplification reagents to the desired target sequence and in addition, favour the performance of post-amplification immunocytochemistry. Following this (if a non-isotopic labelling method is used), a blocking step must be employed, depending on the method used for post-amplification detection of product (e.g. peroxidase or alkaline phosphatase detection systems), in the former, endogenous peroxidase is quenched by incubation in a 3% H202 solution with sodium azide, while 20% ice-cold acetic acid blocks intestinal alkaline phosphatase.
Amplification As in solution phase PCR, successful amplification is governed by: (a) careful optimization of cycling parameters; (b) appropriate design of primer pairs (taking into account their Tm (i.e. specific melting temperature), their ability to form primer-dimers and uniqueness); and (c) optimization of Mg > concentrations, needed to drive the amplification reaction. Due to reagent sequestration (see below), higher concentrations of amplification reagents are required during in situ amplification than for conventional solution phase techniques, including primers, dNTPs and Mg >. Mg concentrations in particular have to be carefully optimized with satisfactory amplification occurring at 2.5-
5.5 mM for most applications. Amplification reagent volumes usually vary between 15 and 75 gl, depending on the surface area of the cell preparation/tissue section used. This is important, as 'patchy' amplification may occur over the surface of the slide because of volume variations consequent to localized amplification failure. When using the Perkin Elmer Gene Amp in situ PCR 1000 system, 25-50 btl volumes are recommended. Initial denaturation of DNA can be achieved before amplification either during permeabilization or following the fixation process. Alternatively, it may be performed at the beginning of the amplification protocol itself. Denaturation can be achieved by heat, heat/ formamide or alkaline denaturation. 5 In addition, most investigators advocate the use of 'hot start' PCR to reduce mispriming and primer oligomerization and Nuovo has suggested the addition of single-strand binding protein (SSB) derived from Escherichia coli. The precise mode of action of this protein is unknown but it functions in DNA replication and repair by preventing primer mispriming and oligomerization. Optimization of cycling parameters has to be performed for each particular assay. As preservation of cellular morphology is paramount, the aim is to achieve optimum amplification at the lowest number of cycles, while at the same time minimizing product diffusion. Most protocols employ 25-30 rounds of amplification exceptionally 50 cycles. Some investigators have performed two successive 30-cycle rounds with the addition of new reagents, including primers and DNA polymerase, between each round. A modification of this method is 'nested' PCR, where internal primers 'nested' within the amplicon produced during the first round, are added. Primer selection has evolved around two basic strategies: single primer pairs or multiple primer pairs with or without complementary tails, a')5~ Multiple primer pairs have been designed to generate longer/ overlapping product with the obvious advantage of localization of amplicons and minimal product diffusion. However, if 'hot-start' PCR is employed, single primer pairs usually suffice. P O S T - A M P L I F I C A T I O N WASHING AND FIXATION Post-fixation with 4% paraformaldehyde and/or ethanol may be employed to maintain localization of amplified product. For PCR-ISH, an oligonucleotide or genomic probe is applied at this stage; to achieve maximum specificity, the probe should hybridize to sequences internal to the amplified product only. However, genomic probes are not restricted to these sequences and appear to provide comparable results. Following in situ amplification, most protocols include a post-amplification washing step (with sodium chloride sodium citrate (SSC), formamide and varying washing temperatures) to remove diffused extracellular product that may result in non-specific staining and generation of false-positive results. The 'stringency' of the wash is defined by the set of conditions employed (i.e. SSC concentration, percentage
206 CURRENTDIAGNOSTICPATHOLOGY formamide used and the washing temperature). The investigator attempts to achieve a washing 'window' where the signal to background noise ratio is maximal. D E T E C T I O N OF AMPLIFIED PRODUCT Non-isotopic labels (e.g. biotin, digoxigenin, fluorescein) are more widely used than isotopic labels and, when used in conjunction with a 'sandwich' immunohistochemical detection technique, appear to provide similar degrees of detection sensitivity. These may vary from one-step to five-step detection systems, as for conventional immunocytochemistry. The product is finally visualized by either a colour reaction (e.g. NBT/BCIP or AEC chromagens) or fluorescence. NUOVO14'15 has described the performance of postamplification conventional immunocytochemistry, the obvious advantage being co-localization of product with the cell of interest, e.g. endothelial cell or macrophage. However, attempts to reproduce this by various groups, including our own, have been disappointing and it is likely that most epitopes do not withstand repetitive thermal cycling.
Reaction, tissue and detection controls It is imperative that an extended panel of controls be performed for each individual assay. These include parallel solution phase PCR, omission of primers and/or Taq DNA polymerase, irrelevant primers and/or probes, known negative controls and reference control genes (Figs 4 & 5). The following are the minimal controls required in the PCR-ISH:
9 Reference control gene, e.g. [3-globin or pyruvate
dehydrogenase (PDH) DNase digestion RNase digestion Target primers with irrelevant probe Irrelevant primers with target probe Irrelevant primers with irrelevant probe Reference control gene primers with the target probe Target primer one only Target primer two only No Taq No primers Omit the reverse transcriptase step in RT IS-PCR or RT PCR-ISH 9 In situ hybridization controls for PCR-ISH and RT PCR-ISH 9 Detection controls for immunocytochemical detection systems.
9 9 9 9 9 9 9 9 9 9 9
For in situ PCR, controls a, b, c, h, i, j, k, together with a set-up including one target primer and one irrelevant primer pair are used. Reference control genes, including the use of a single copy mammalian gene such as PDH, are important to assess the degree of amplification in the tissue section/ cell preparation. When amplifying DNA targets, the addition of DNase should abolish the signal. If this does not occur, the signal may have resulted from spurious amplification or alternatively may represent either RNA/cDNA. RNase pre-treatment is mandatory in the assessment of RNA targets (in situ reverse transcriptase [RT] PCR) and should be included in the amplification of DNA templates to minimize false-positive signals originating from cellular RNA. The use of a reference control gene primer pair in
Fig. 4---In situ amplification and NISH of HPV 16 in SiHa cells. (A) NISH showing two copies of HPV 16 in the nucleus. This signal is achieved using a complex immunocytochemical technique. (B) PCRISH of fixed SiHa cell showing two copies of HPV 16 normally present in SiHa cells. 49 (C) Two loci of amplification within a SiHa cell nucleus, using PCR-ISH.49 (D) Over-amplification of HPV 16 E6 genome in SiHa cells. Note that all cells are positive, but nuclear and cytoplasmic positivity seen. (E) Labelled primer driven in situ amplification for E6 HPV 16 region in SiHa cells. (F) Negative control (E)..
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conjunction with target specific probe assesses the degree of 'stickiness' of the target probe sequence and the creation of a false-positive result. The addition of only one primer in the amplification mix generates an 'asymmetric PCR' with a quantative reduction in the amount of product synthesized. Irrelevant primers with irrelevant probe should not generate a signal. The specificity of the in situ hybridization component of PCR-ISH is assessed by employing an irrelevant probe with target-specific primers. The role of primer-primer dimerization and primer oligomerization in the generation of false-positive signals is assessed by excluding Taq DNA polymerase, whereas the contribution of non-specific elongation of nicked DNA in tissue sections is examined by the exclusion of primers. The latter is an extremely important control for ISH-PCR. In the assessment of RT-IS PCR, omission of the reverse transcriptase step is important to ensure adequate DNAase digestion. As in routine in situ hybridization, hybridization controls and detection controls are essential to exclude false positive/negative results due to failure of the ISH step or aberrant staining of tissues by the detection system.
reasonably simple, using currently available chemistries. A cDNA template is first created using a reverse transcriptase enzyme, usually Moloney mouse leukaemia virus (MMLV) RT and then followed by amplification of the newly synthesized cDNA template. A labelled nucleotide (e.g. biotin 11 dUTP) or a labelled primer can be used (RT IS-PCR). Alternatively, a post-amplification in situ hybridization step may be employed (RT PCR-ISH). The above techniques employ a two-step approach, i.e. reverse transcription and then amplification. We have now described a single step methodology using the rTth DNA polymerase enzyme that obviates the need for splitting the reaction, rTth polymerase possesses both reverse transcriptase and DNA polymerase activity, and is obviously suited for in situ amplification applications.
AMPLIFICATION OF RNA TARGETS IN CELLS AND TISSUES
Cell and tissue preparation The same basic principles apply as for DNA in situ amplification. The techniques specifically designed for amplification of RNA targets are RT IS-PCR and RT PCR-ISH. In general, RNA targets are easier to amplify than DNA targets, because of the increased number of starting copies of target. However, many variables outside the control of the operator interact with RNA templates in cells and tissue sections, which in many cases jeopardizes the integrity of RNA. Once cells or tissues are removed from the body, RNA degradation begins almost instantaneously. Indeed the environment is rich in RNases: fingers, gloves, bench tops are just a few of the many sources. Fixative solutions contain specific RNases that degrade RNA, and tissue processing, again contaminated by RNases, minimizes the amount of target RNA that can be amplified. Ideally, an RNase-free working environment should be created for RNA in situ amplification. For optimal preservation of RNA in tissue sections, immediate fixation in RNase-free solutions should be carried out. Alcohol, acetic acid:alcohol and neutral buffered formaldehyde fixatives made up in autoclaved DEPC (di-ethylpyrocarbonate)-treated water should be used. Again, all protocols for umnasking of nucleic acid should employ RNase-free conditions where possible.
Amplification, methodology and chemistries The amplification of RNA targets in cells and tissue is
I
I=i9.5--(A) Pyruvate dehydrogenase (PDH) DNA amplification in CaSki cells using labelled primer driven in situ amplification. Note the localization of the signal to the nucleus of the cell. (B) RT IS-PCR for localization of PDH mRNA in CaSki cells. Note localization of amplified product in the cytoplasmic compartment of the cell. (C) Negative control omitting the reverse transcriptase (RT) step.
208 CURRENTDIAGNOSTICPATHOLOGY
Controls for RNA in situ amplification In general, the same controls apply as for DNA in situ amplification. Omission of the reverse transcriptase step will obviously yield a faint or negative result depending on whether RT IS-PCR or RT PCR-ISH is used. Of importance for RNA assays is the optimal digestion of DNA in cells and tissue sections, which in many cases can be difficult to remove.
PROBLEMS ASSOCIATED WITH THE TECHNIQUES Many groups have encountered problems with the performance of in situ PCR and as yet no universally applicable technique is available. 49'5~ This is influenced by a number of factors, including the starting materials, fixation conditions and target. In situ PCR is particularly fraught with difficulties, especially with paraffin-embedded material where non-specific incorporation of labelled nucleotide may occur in the presence of Taq DNA polymerase. PCR-ISH appears to be more specific, especially if a 'hot-start' modification is employed or alternatively, if multiple primer pairs are used. PCR-ISH protocols, in general, are more sensitive but, in contrast to solution phase PCR, are less efficient with apparent linear amplification only. The degree of amplification is difficult to assess and contradictory estimates have been documented in the literature. N u o v o 12-17 has reported a 200-300-fold increase in product, in contrast to Embretson and colleagues 3~who estimate an increase of 10-30 fold only. Our experience would tend to support the latter figure. The major limitation with IS-PCR, as previously mentioned, is the non-specific incorporation of nucleotides into damaged DNA by Taq DNA p01ymerase. This is cycle- and DNA polymerase-dependent and may occur in the absence of primers and/or with a 'hot-start' modification. Therefore, the routine use of in situ PCR is as yet not feasible because of the risk of generating false signals. However, Gosden 33 has reported the use of strand break joining in chromosomal work to eliminate spurious incorporation during in situ PCR. Pre-treatment with di-deoxy blockage has also been documented to eliminate non-specific incorporation, but this is not always successful. Another approach has been strand 'super-denaturation', i.e. dsDNA is denatured at high temperatures. The DNA is then maintained in a denatured state for an extended period of time (5-10 min) but again, this has produced inconsistent results. We routinely employ 'super-denaturation' prior to performing conventional 'hot start' PCR at 70~ (O'Leary, 1996, submitted for publication).
Sequestration of reagents As alluded to previously, in contrast to solution phase PCR, increased concentrations of reagents axe required for successful in situ amplification (usually of the order of two to five times). This is thought to be due to reagent
sequestration as a result of reagents adhering to slides or to the coating materials used. An additional possibility is that the reagents may intercalate with fixative residues left in tissues. However, it has been documented that pre-treating slides with 0.1-1% bovine serum albumin (BSA) allows a reduction in reagent concentration, which may function by blocking this sequestration. 5~
Diffusion of amplicons and back diffusion An inevitable consequence of in situ amplification is product diffusion from the site of synthesis, which may occur as a result of perrneabilization and/or cell truncation (see below). One approach is to reduce the number of cycles. Another frequently employed strategy is to post-fix the slides in ethanol or paraformaldehyde, which help to maintain localization of product. Alternative approaches include overlaying the tissue section with agarose and/or incorporation of biotin-substituted nucleotides (analogous to in situ PCR). This latter modification promotes the generation of bulkier products which are less likely to diffuse.
Patchy amplification Invariably with any in situ amplification technique, some degree of patchy amplification will occur, with between 30% and 80% of cells containing the target sequence of interest staining at any one time. There are many reasons for this patchy amplification; these include non-uniform digestion with variations in cell permeability, failure to completely disassociate DNA-histone protein crosslinkages and thus to interfere with DNA polymerase progression along the template, and cell truncation. This latter factor is an inevitable consequence of microtome sectioning where cell 'semi-spheres' are created. As a result, the nuclear contents are truncated giving rise to two possibilities: the desired target sequence may not be present or the target sequence may be present but the product may have diffused out. CURRENT APPLICATIONS In situ amplification techniques and protocols are, on
the whole, still being developed. Several groups have documented their success with the identification of single copy sequences in cells and low copy number DNA sequences in tissue sections, including viral DNA sequences (e.g. HIV, HPV, MMTV provirus, CMV, HBV, KSHV) especially in latent infection (Table 1). Endogenous human DNA sequences have also been examined, including single copy human genes, chromosomal re-arrangements and translocations (Table 1). In situ amplification techniques have exciting potential, particularly with regard to the amplification of turnout-specific sequences, including translocations (e.g. t-11:22 in PNET), T-cell receptor gene re-arrangements and point mutations. As general expertise with these techniques improves, the potential in terms of both research and clinical applications is enormous (see Table 2).
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Table 1--Targets amplified by investigators using in situ amplification Viruses (DNA and RNA) 9 HIV-1 (mononuclear cells, sperm, fixed brain tissue, macrophages, oral mucosal epithelial cells) 3'<7'252~ 9 HPV 6,11, 16,18, etc. (cervical biopsies, cell lines) l~j6,w'44 9 HBV, HCV (liver biopsies) ~4'4~ 9 CMV (archival paraffin-embedded tissues)z4 9 HHV-6, HHV-8/KSHV (Kaposi's sarcoma, lymphomas, etc.) z~ 9 HSV-DNA/RNA (trigeminal ganglia, disseminated encephalitis) 26,34,35 9 LGV (lymphogranuloma venerum) (Bagasra et al.) 51 Oncogene/tumour suppressor genes 9 p53 mutations (paraffin-embedded tissue) (Bagasra et al.) 5~ 9 Gene re-arrangements (t11:22 in PNET, t14:18 in follicular lymphoma)41 9 ras mutations (H, Ki, N-ras, codon 12, 13 and 61) 9 Chromosome mapping (PRINS and cycling PRINS) 33 9 T-cell receptor gene re-arrangements and immunoglobulin heavy and light V chains3~ Growth factors, markers of malignancy and other biological markers 9 Metalloproteinases and their inhibitors ]7 9 EGF receptor mRNA expression3s 9 Endothelial receptor mRNA 29 9 Nitric oxide synthase in multiple sclerosis5
Table 2--Potential uses of in situ amplification techniques in pathology Detection of viral genes in tissues, e.g. HPV, EBV, CMV, KSHV, HIV 1 and 2 Detection of bacterial species, e.g. Mycobacterium tuberculosis, M. leprae, M. bovis, etc. Detection of mammalian structural genes in embryogenesis and dysmorphogcnesis Detection of point mutations in oncogenes and turnout suppressor genes, e.g. ras, p53 Single cell allelic discriminatkm and individual cell haplotyping, e.g. cystic fibrosis, thalassaemias, etc. Detectkm of chromosomal translocations, e.g. t l 1:22, t14:18, etc. Quantitative in situ amplification for viral load and oncogene expression (IS-TaqMAN PCR) Assessment of loss of heterozygosity (LOH) and allelic imbahmce Forensic identification of tissues, e.g. HLA DQ, etc
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Mini-symposium: Advances in diagnostic technology
In situ amplification: its application to
diagnostic pathology
J. J. O'Leary, M. M. Kennedy, R. J. Landers and J. O'D. McGee
initially described technique of in situ PCR, to include PRINS (primed in situ labelling), cycling PRINS, in situ PNA PCR (peptide nucleic acid PCR), in situ PNA PCR, IS-Taq Man PCR and allele specific amplification.
INTRODUCTION Solution phase polymerase chain reaction (PCR) has become one of the most revolutionary tools in molecular pathology. The technique, conventionally performed in a tube, permits the amplification of single copy mammalian/viral targets (either DNA or RNA) in fresh/frozen and archival paraffin-embedded material. However, the inability to visualize and localize amplified product within cells and tissue specimens has been a major limitation, especially for pathologists attempting to correlate genetic events with pathological changes. In situ hybridization (ISH) has partly addressed this problem by permitting localization of specific nucleic acid sequences at the individual cell level. However, most conventional non-isotopic in situ detection systems do not detect single copy genes, except for those incorporating elaborate sandwich detection techniques as described in 1992 by Herrington.l Before amplification can be performed in solution phase PCR (SPPCR), nucleic acid extraction is firstly carried out, which necessitates cellular destruction. Subsequent correlation of results with histological features is consequently not possible. Recently, a number of studies have described 'hybrid' techniques coupling PCR with in situ hybridization. 2-~4 The techniques have not been universally accepted because of technological problems encountered during amplification of the desired nucleic acid sequence. However, in recent months, dedicated equipment and chemistry have greatly facilitated the performance of in situ amplification and are rapidly expanding the potential use of these tools in diagnostic pathology. The repertoire of in situ amplification has now been extended to include a number of modifications of the
GENERAL
PRINCIPLES
All of the above techniques attempt to create doublestranded or single-stranded DNA amplicons within the cell, which can either be detected directly or following an in situ hybridization step. For successful application of the techniques, the aim is to achieve a fine balance between adequate digestion of cells (allowing access of amplification reagents) and maintaining localization of amplified product within the cellular compartment while preserving tissue/cell morphology. To do this, the techniques require specific cyclical thermal changes to occur at the individual cell level, akin to that which occurs in solution phase PCR. These changes initially bring about denaturation of double-stranded DNA (dsDNA) to single-stranded form (ssDNA), if a DNA target is being amplified. For RNA target specific amplification, the RNA template is already single stranded and reverse transcription is carried out to create a cDNA template. Specific short runs of oligonucleotides (primers) are then annealed to the respective ends of the desired target sequence and a thermostable enzyme (Taq DNA polymerase or Klenow fragment) is used to extend or ligate (DNA ligase, in IS-LCR; see below) the correctly positioned primers. Subsequent rounds of thermocycling then increase the copy number of the desired target sequence, in a nucleic acid amplification reaction. However, unlike solution phase PCR, an exponential increase in the amount of amplified product is never achieved. Instead, linear amplification occurs in most situations because of the relative inefficiency of the techniques; because of the compactness of the nuclear compartment of the cell
J. J. O'Leary, M. M. Kennedy, R. J. Landers and J. O'D. McGee,
Nuffield Departmentof Pathologyand Bacteriology,Universityof Oxford, OxfordOX3 9DU, UK 201
202 CURRENTDIAGNOSTICPATHOLOGY (containing dsDNA ssDNA pre-mRNA and histone proteins), there are problems of accessibility of amplification reagents to the desired nucleic acid sequence. Once the amplicon is created, detection must then be carried out. If the investigator has utilized a labelled primer or nucleotide, then the amplicon is labelled and can be demonstrated directly by immunocytochemical techniques. Alternatively, an in situ hybridization step (with a single-stranded oligoprobe or a double-stranded genomic probe) is carried out post-amplification, adhering to the general rules of standard in situ hybridization. Definitions Several essentially similar techniques are now described as follows.
DNA in situ PCR (IS-PCR) PCR amplification of cellular DNA sequences in tissue specimens using a labelled nucleotide (i.e. dUTP) within the PCR reaction mix. The labelled product is then detected by standard detection techniques as for conventional in situ hybridization or immunocytochemistry (Fig. 1).
Labelled primer driven in situ amplification (LPDISA) In situ amplification of DNA sequences using a labelled primer within the PCR reaction mix. The labelled product is then detected as for DNA IS-PCR. PCR in situ hybridization (PCR-ISH) PCR amplification of cellular DNA sequences in tissue specimens followed by in situ hybridization detection of
the amplified product with a labelled internal or genomic probe. The labels used can either be isotopic (p3z, $35) or non-isotopic (e.g. biotin, digoxigenin, fluorescein) (Fig. 1).
Reverse transcriptase in situ PCR (RT in situ PCR) The amplification of mRNA sequences in cells and tissue specimens by firstly creating a copy DNA template (cDNA) with RT and then amplifying the newly created DNA template as for DNA IS-PCR.
Reverse transcriptase PCR in situ hybridization (RT PCR-ISH) Amplification of RNA sequences in cell and tissue specimens by creating a cDNA template with RT. The newly created cDNA is then amplified, and the amplicon probed with an internal oligonucleotide as in PCR-ISH.
PRINS (primed in situ amplification) and cycling PRINS Amplification of specific genetic sequences in metaphase chromosome spreads or interphase nuclei, using one primer to generate single-stranded PCR product. If many rounds of amplification are utilized then the technique is called cycling PRINS (Fig. 2A and B). 33 In situ peptide nucleic acid PCR (IS-PNA-PCR) and PCR-PNA-ISH
IS-PNA-PCR. The amplification of DNA targets using a DNA mimic molecule, PNA. PNA is a simple molecule, made up of repeating N-(2-aminoethyl)-glycine units linked by amide bonds. Purine (A and G) and pyrimidine
Fig, 1--Schematic representation of in situ PCR and PCR-ISH.
IN S1TUAMPLIFICATION 203
Fig. 2--Schematic representation of (A) PRINS and (B) cycling PRINS by a single primer approach.
bases (C and T) are attached to the back bone by methylene carbonyl linkages. PNA is not elongated by Taq DNA polymerase when used as a primer and thereforc can be used in primer exclusion assays, which allows the discrimination of point mutations and direct individual cell haplotyping. PCR-PNA-ISH. In this second reaction which employs PNA, a 15-20 mer PNA probe is used for the in situ
hybridization step following amplification. PNAs have higher melting temperatures (Tms) than DNA oligoprobes and single point mutations in a PNA-DNA duplex lower the Tm by approximately 15~ as compared to the corresponding DNA-DNA mismatch duplex. 45 IS-TaqMan PCR
Amplification of DNA sequences using a conventional primer pair as in standard PCR. However, an internal TaqMan probe is added to the amplification mix. A flourescent reporter molecule (FAM, HEX, etc.) is placed at the 5' end of the probe. At the 3' end, a quencher molecule (again fluorescent, usually TAMRA) is positioned. Once the probe is linearized and intact, the proximity of the quencher to the reporter molecule does not allow any fluorescence from the reporter molecule.
Tat} DNA polymerase possesses two properties important for the reaction: (1) Y-strand fork displacement (which allows Taq DNA polymerase to lift off a single strand in Y configuration and (2) 5'-3" endonucleolytic activity, which causes cleavage of the linker arm which attaches the reporter molecule to the 5' end of the Taq Man probe (Fig. 3), thereby giving rise to fluorescence if specific amplification has o c c u r r e d . 46'47 In situ allele specff)'c amplification In situ allele specific amplification (IS-ASA) technique
utilizes ARMS PCR (amplification refi'actory mutation system) which has the ability to detect point polymorphisms in human DNA sequences using artificially created base pair mismatches at the 3' end of PCR primers. If the polymorphism matching that of the primer sequence is present, amplification of that sequence wilt preferentially occur.
AMPLIFICATION OF DNA TARGETS IN CELLS AND TISSUES Equipment Several different types of equipment can be used for in situ amplification, ranging from standard thermal cycters
204 CURRENTDIAGNOSTICPATHOLOGY
Fig. 3--Schematic representation of Taq Man PCR (see text for details of method).
(with modifications), thermocycling ovens and specifically dedicated thermocyclers (Perkin Elmer Gene Amp in situ PCR system 1000, Hybrid Omnigene/Omnislide and MJB slide thermocycler). If a standard thermal cycler is used, then an amplification chamber initially must be created for the slide. This is usually made from aluminium (aluminium foil boat). This boat containing the slide is placed on the thermal cycler, covered with mineral oil and then wrapped completely. However, optimization of thermal conduction is never completely achieved. 'Thermal lag' (i.e. differences in temperature between the block face, the glass slide and the PCR reaction mix at each temperature step of~ the reaction cycle) is commonly encountered. 19 This has been addressed by the newer in situ amplification machines, which offer in-built slide temperature calibration curves with greater thermodynamic control.
Starting material In 1990, Haase initially described in situ PCR in intact fixed single cells, suspended in PCR reaction buffer. After amplification, cells were cytocentrifuged on to glass slides and the amplified product was detected by in situ hybridization. 2 Other early investigators used pieces of glass slides (with cells from cytocentrifuge preparations) in standard ependorf tubes, incubated directly in PCR reaction buffer. 1~ More recently techniques using tissues and cells attached to microscope slides have been described with amplification carried out either on heating blocks or in cycling ovens. 3~4 For IS-PCR, PCR-ISH, IS-LCR, IS-PNA PCR and IS-TaqMAN PCR, fixed cells and tissues - including archival paraffin-embedded material - can be used for
amplification. Best results are obtained with freshly fixed cells and tissues, although successful amplification with old archival material (up to 40 years) has been achieved (O. Bagasra, personal communication). Fixed metaphase chromosome spreads and interphase nuclei can be used for the detection of specific subchromosomal regions using PRINS and cycling PRINS. 33 For successful in situ amplification to occur, a rigid cellular cytoskeleton must be created which provides a suitable micro-environment allowing access of amplification reagents with minimal leakage of amplified product. Satisfactory results may be obtained with tissues fixed in 1-4% paraformaldehyde, neutral buffered formaldehyde (NBF) and 10% formalin (12-24h for biopsy/solid tissue; 10-30 min for cytological preparations). 16J8 Less consistent results are obtained with ethanol and acetic acid fixed t i s s u e s . 16 Fixation of cells with formaldehyde fixatives produces a number of drawbacks. Formaldehyde is not easily removed from tissues, even after tissue processing. Aldehyde groups react with DNA and histone proteins to form DNA-DNA and DNA-histone protein cross-links. 48 Formaldehyde fixation also 'nicks' DNA template (random breaks in dsDNA), which may be non-blunt ended (i.e. non-overlapping ends). These nicks may subsequently act as potential priming sites for Taq DNA polymerase leading to incorporation and elongation of labelled and unlabelled nucleotides (i.e. dATP, etc.) analogous to in situ end labelling for apoptosis. This process occurs at room temperature, leading to spurious results with DNA IS-PCR. Because repeated cycles of heating and cooling are used during in situ amplification, it is imperative that cells and tissues are adequately attached to a solid support (usually glass), so that detachment does not
IN SITU AMPLIFICATION 205
occur. Glass slides are pre-treated with coating agents to ensure maximal section adhesion, the most commonly used agents being aminopropyltriethoxysilane (APES), Denhardt's solution and Elmer's glue.
Cell and tissue permeabilization Cells must be adequately digested and permeabilized to facilitate access of reagents. This is usually achieved by protease treatment (e.g. proteinase K, pepsin or trypsin) and/or mild acid hydrolysis (0.01 N-0.1 N HC1). While extended proteolytic digestion is performed for solution phase PCR (often up to 24--48 h) to overcome DNAhistone protein cross-linking, digestion of cells and tissues for in situ amplification is limited by the necessity of maintaining cell morphology, architecture and cell/ tissue adhesion. Maximal digestion times and protease concentrations have to be optimized for the particular tissue/cytological preparation employed. Usual working conditions at 37~ are as follows: 0.1 mg/ml proteinase K for 15-25 rain for fixed tissues; 0.01-0.1 mg/ml proteinase K for up to 5 rain for cytological preparations. Longer digestion times inevitably compromise cellular morphology, but as a result incomplete dissociation of histone protein-DNA cross-links occurs, hindering the progression of Taq DNA polymerase along the native DNA template. Acid hydrolysis probably acts by driving such cross-links to complete dissociation. Alternatively, microwave irradiation of cells and tissue sections can be used to expose nucleic acid templates. Short pulses of microwave irradiation (with or without proteolysis) with a citrate buffer analogous to antigen retrieval for immunocytochemistry - effectively allow access of amplification reagents to the desired target sequence and in addition, favour the performance of post-amplification immunocytochemistry. Following this (if a non-isotopic labelling method is used), a blocking step must be employed, depending on the method used for post-amplification detection of product (e.g. peroxidase or alkaline phosphatase detection systems), in the former, endogenous peroxidase is quenched by incubation in a 3% H202 solution with sodium azide, while 20% ice-cold acetic acid blocks intestinal alkaline phosphatase.
Amplification As in solution phase PCR, successful amplification is governed by: (a) careful optimization of cycling parameters; (b) appropriate design of primer pairs (taking into account their Tm (i.e. specific melting temperature), their ability to form primer-dimers and uniqueness); and (c) optimization of Mg > concentrations, needed to drive the amplification reaction. Due to reagent sequestration (see below), higher concentrations of amplification reagents are required during in situ amplification than for conventional solution phase techniques, including primers, dNTPs and Mg >. Mg concentrations in particular have to be carefully optimized with satisfactory amplification occurring at 2.5-
5.5 mM for most applications. Amplification reagent volumes usually vary between 15 and 75 gl, depending on the surface area of the cell preparation/tissue section used. This is important, as 'patchy' amplification may occur over the surface of the slide because of volume variations consequent to localized amplification failure. When using the Perkin Elmer Gene Amp in situ PCR 1000 system, 25-50 btl volumes are recommended. Initial denaturation of DNA can be achieved before amplification either during permeabilization or following the fixation process. Alternatively, it may be performed at the beginning of the amplification protocol itself. Denaturation can be achieved by heat, heat/ formamide or alkaline denaturation. 5 In addition, most investigators advocate the use of 'hot start' PCR to reduce mispriming and primer oligomerization and Nuovo has suggested the addition of single-strand binding protein (SSB) derived from Escherichia coli. The precise mode of action of this protein is unknown but it functions in DNA replication and repair by preventing primer mispriming and oligomerization. Optimization of cycling parameters has to be performed for each particular assay. As preservation of cellular morphology is paramount, the aim is to achieve optimum amplification at the lowest number of cycles, while at the same time minimizing product diffusion. Most protocols employ 25-30 rounds of amplification exceptionally 50 cycles. Some investigators have performed two successive 30-cycle rounds with the addition of new reagents, including primers and DNA polymerase, between each round. A modification of this method is 'nested' PCR, where internal primers 'nested' within the amplicon produced during the first round, are added. Primer selection has evolved around two basic strategies: single primer pairs or multiple primer pairs with or without complementary tails, a')5~ Multiple primer pairs have been designed to generate longer/ overlapping product with the obvious advantage of localization of amplicons and minimal product diffusion. However, if 'hot-start' PCR is employed, single primer pairs usually suffice. P O S T - A M P L I F I C A T I O N WASHING AND FIXATION Post-fixation with 4% paraformaldehyde and/or ethanol may be employed to maintain localization of amplified product. For PCR-ISH, an oligonucleotide or genomic probe is applied at this stage; to achieve maximum specificity, the probe should hybridize to sequences internal to the amplified product only. However, genomic probes are not restricted to these sequences and appear to provide comparable results. Following in situ amplification, most protocols include a post-amplification washing step (with sodium chloride sodium citrate (SSC), formamide and varying washing temperatures) to remove diffused extracellular product that may result in non-specific staining and generation of false-positive results. The 'stringency' of the wash is defined by the set of conditions employed (i.e. SSC concentration, percentage
206 CURRENTDIAGNOSTICPATHOLOGY formamide used and the washing temperature). The investigator attempts to achieve a washing 'window' where the signal to background noise ratio is maximal. D E T E C T I O N OF AMPLIFIED PRODUCT Non-isotopic labels (e.g. biotin, digoxigenin, fluorescein) are more widely used than isotopic labels and, when used in conjunction with a 'sandwich' immunohistochemical detection technique, appear to provide similar degrees of detection sensitivity. These may vary from one-step to five-step detection systems, as for conventional immunocytochemistry. The product is finally visualized by either a colour reaction (e.g. NBT/BCIP or AEC chromagens) or fluorescence. NUOVO14'15 has described the performance of postamplification conventional immunocytochemistry, the obvious advantage being co-localization of product with the cell of interest, e.g. endothelial cell or macrophage. However, attempts to reproduce this by various groups, including our own, have been disappointing and it is likely that most epitopes do not withstand repetitive thermal cycling.
Reaction, tissue and detection controls It is imperative that an extended panel of controls be performed for each individual assay. These include parallel solution phase PCR, omission of primers and/or Taq DNA polymerase, irrelevant primers and/or probes, known negative controls and reference control genes (Figs 4 & 5). The following are the minimal controls required in the PCR-ISH:
9 Reference control gene, e.g. [3-globin or pyruvate
dehydrogenase (PDH) DNase digestion RNase digestion Target primers with irrelevant probe Irrelevant primers with target probe Irrelevant primers with irrelevant probe Reference control gene primers with the target probe Target primer one only Target primer two only No Taq No primers Omit the reverse transcriptase step in RT IS-PCR or RT PCR-ISH 9 In situ hybridization controls for PCR-ISH and RT PCR-ISH 9 Detection controls for immunocytochemical detection systems.
9 9 9 9 9 9 9 9 9 9 9
For in situ PCR, controls a, b, c, h, i, j, k, together with a set-up including one target primer and one irrelevant primer pair are used. Reference control genes, including the use of a single copy mammalian gene such as PDH, are important to assess the degree of amplification in the tissue section/ cell preparation. When amplifying DNA targets, the addition of DNase should abolish the signal. If this does not occur, the signal may have resulted from spurious amplification or alternatively may represent either RNA/cDNA. RNase pre-treatment is mandatory in the assessment of RNA targets (in situ reverse transcriptase [RT] PCR) and should be included in the amplification of DNA templates to minimize false-positive signals originating from cellular RNA. The use of a reference control gene primer pair in
Fig. 4---In situ amplification and NISH of HPV 16 in SiHa cells. (A) NISH showing two copies of HPV 16 in the nucleus. This signal is achieved using a complex immunocytochemical technique. (B) PCRISH of fixed SiHa cell showing two copies of HPV 16 normally present in SiHa cells. 49 (C) Two loci of amplification within a SiHa cell nucleus, using PCR-ISH.49 (D) Over-amplification of HPV 16 E6 genome in SiHa cells. Note that all cells are positive, but nuclear and cytoplasmic positivity seen. (E) Labelled primer driven in situ amplification for E6 HPV 16 region in SiHa cells. (F) Negative control (E)..
IN SITU AMPLIFICATION 207
conjunction with target specific probe assesses the degree of 'stickiness' of the target probe sequence and the creation of a false-positive result. The addition of only one primer in the amplification mix generates an 'asymmetric PCR' with a quantative reduction in the amount of product synthesized. Irrelevant primers with irrelevant probe should not generate a signal. The specificity of the in situ hybridization component of PCR-ISH is assessed by employing an irrelevant probe with target-specific primers. The role of primer-primer dimerization and primer oligomerization in the generation of false-positive signals is assessed by excluding Taq DNA polymerase, whereas the contribution of non-specific elongation of nicked DNA in tissue sections is examined by the exclusion of primers. The latter is an extremely important control for ISH-PCR. In the assessment of RT-IS PCR, omission of the reverse transcriptase step is important to ensure adequate DNAase digestion. As in routine in situ hybridization, hybridization controls and detection controls are essential to exclude false positive/negative results due to failure of the ISH step or aberrant staining of tissues by the detection system.
reasonably simple, using currently available chemistries. A cDNA template is first created using a reverse transcriptase enzyme, usually Moloney mouse leukaemia virus (MMLV) RT and then followed by amplification of the newly synthesized cDNA template. A labelled nucleotide (e.g. biotin 11 dUTP) or a labelled primer can be used (RT IS-PCR). Alternatively, a post-amplification in situ hybridization step may be employed (RT PCR-ISH). The above techniques employ a two-step approach, i.e. reverse transcription and then amplification. We have now described a single step methodology using the rTth DNA polymerase enzyme that obviates the need for splitting the reaction, rTth polymerase possesses both reverse transcriptase and DNA polymerase activity, and is obviously suited for in situ amplification applications.
AMPLIFICATION OF RNA TARGETS IN CELLS AND TISSUES
Cell and tissue preparation The same basic principles apply as for DNA in situ amplification. The techniques specifically designed for amplification of RNA targets are RT IS-PCR and RT PCR-ISH. In general, RNA targets are easier to amplify than DNA targets, because of the increased number of starting copies of target. However, many variables outside the control of the operator interact with RNA templates in cells and tissue sections, which in many cases jeopardizes the integrity of RNA. Once cells or tissues are removed from the body, RNA degradation begins almost instantaneously. Indeed the environment is rich in RNases: fingers, gloves, bench tops are just a few of the many sources. Fixative solutions contain specific RNases that degrade RNA, and tissue processing, again contaminated by RNases, minimizes the amount of target RNA that can be amplified. Ideally, an RNase-free working environment should be created for RNA in situ amplification. For optimal preservation of RNA in tissue sections, immediate fixation in RNase-free solutions should be carried out. Alcohol, acetic acid:alcohol and neutral buffered formaldehyde fixatives made up in autoclaved DEPC (di-ethylpyrocarbonate)-treated water should be used. Again, all protocols for umnasking of nucleic acid should employ RNase-free conditions where possible.
Amplification, methodology and chemistries The amplification of RNA targets in cells and tissue is
I
I=i9.5--(A) Pyruvate dehydrogenase (PDH) DNA amplification in CaSki cells using labelled primer driven in situ amplification. Note the localization of the signal to the nucleus of the cell. (B) RT IS-PCR for localization of PDH mRNA in CaSki cells. Note localization of amplified product in the cytoplasmic compartment of the cell. (C) Negative control omitting the reverse transcriptase (RT) step.
208 CURRENTDIAGNOSTICPATHOLOGY
Controls for RNA in situ amplification In general, the same controls apply as for DNA in situ amplification. Omission of the reverse transcriptase step will obviously yield a faint or negative result depending on whether RT IS-PCR or RT PCR-ISH is used. Of importance for RNA assays is the optimal digestion of DNA in cells and tissue sections, which in many cases can be difficult to remove.
PROBLEMS ASSOCIATED WITH THE TECHNIQUES Many groups have encountered problems with the performance of in situ PCR and as yet no universally applicable technique is available. 49'5~ This is influenced by a number of factors, including the starting materials, fixation conditions and target. In situ PCR is particularly fraught with difficulties, especially with paraffin-embedded material where non-specific incorporation of labelled nucleotide may occur in the presence of Taq DNA polymerase. PCR-ISH appears to be more specific, especially if a 'hot-start' modification is employed or alternatively, if multiple primer pairs are used. PCR-ISH protocols, in general, are more sensitive but, in contrast to solution phase PCR, are less efficient with apparent linear amplification only. The degree of amplification is difficult to assess and contradictory estimates have been documented in the literature. N u o v o 12-17 has reported a 200-300-fold increase in product, in contrast to Embretson and colleagues 3~who estimate an increase of 10-30 fold only. Our experience would tend to support the latter figure. The major limitation with IS-PCR, as previously mentioned, is the non-specific incorporation of nucleotides into damaged DNA by Taq DNA p01ymerase. This is cycle- and DNA polymerase-dependent and may occur in the absence of primers and/or with a 'hot-start' modification. Therefore, the routine use of in situ PCR is as yet not feasible because of the risk of generating false signals. However, Gosden 33 has reported the use of strand break joining in chromosomal work to eliminate spurious incorporation during in situ PCR. Pre-treatment with di-deoxy blockage has also been documented to eliminate non-specific incorporation, but this is not always successful. Another approach has been strand 'super-denaturation', i.e. dsDNA is denatured at high temperatures. The DNA is then maintained in a denatured state for an extended period of time (5-10 min) but again, this has produced inconsistent results. We routinely employ 'super-denaturation' prior to performing conventional 'hot start' PCR at 70~ (O'Leary, 1996, submitted for publication).
Sequestration of reagents As alluded to previously, in contrast to solution phase PCR, increased concentrations of reagents axe required for successful in situ amplification (usually of the order of two to five times). This is thought to be due to reagent
sequestration as a result of reagents adhering to slides or to the coating materials used. An additional possibility is that the reagents may intercalate with fixative residues left in tissues. However, it has been documented that pre-treating slides with 0.1-1% bovine serum albumin (BSA) allows a reduction in reagent concentration, which may function by blocking this sequestration. 5~
Diffusion of amplicons and back diffusion An inevitable consequence of in situ amplification is product diffusion from the site of synthesis, which may occur as a result of perrneabilization and/or cell truncation (see below). One approach is to reduce the number of cycles. Another frequently employed strategy is to post-fix the slides in ethanol or paraformaldehyde, which help to maintain localization of product. Alternative approaches include overlaying the tissue section with agarose and/or incorporation of biotin-substituted nucleotides (analogous to in situ PCR). This latter modification promotes the generation of bulkier products which are less likely to diffuse.
Patchy amplification Invariably with any in situ amplification technique, some degree of patchy amplification will occur, with between 30% and 80% of cells containing the target sequence of interest staining at any one time. There are many reasons for this patchy amplification; these include non-uniform digestion with variations in cell permeability, failure to completely disassociate DNA-histone protein crosslinkages and thus to interfere with DNA polymerase progression along the template, and cell truncation. This latter factor is an inevitable consequence of microtome sectioning where cell 'semi-spheres' are created. As a result, the nuclear contents are truncated giving rise to two possibilities: the desired target sequence may not be present or the target sequence may be present but the product may have diffused out. CURRENT APPLICATIONS In situ amplification techniques and protocols are, on
the whole, still being developed. Several groups have documented their success with the identification of single copy sequences in cells and low copy number DNA sequences in tissue sections, including viral DNA sequences (e.g. HIV, HPV, MMTV provirus, CMV, HBV, KSHV) especially in latent infection (Table 1). Endogenous human DNA sequences have also been examined, including single copy human genes, chromosomal re-arrangements and translocations (Table 1). In situ amplification techniques have exciting potential, particularly with regard to the amplification of turnout-specific sequences, including translocations (e.g. t-11:22 in PNET), T-cell receptor gene re-arrangements and point mutations. As general expertise with these techniques improves, the potential in terms of both research and clinical applications is enormous (see Table 2).
IN SITU AMPLIFICATION
Table 1--Targets amplified by investigators using in situ amplification Viruses (DNA and RNA) 9 HIV-1 (mononuclear cells, sperm, fixed brain tissue, macrophages, oral mucosal epithelial cells) 3'<7'252~ 9 HPV 6,11, 16,18, etc. (cervical biopsies, cell lines) l~j6,w'44 9 HBV, HCV (liver biopsies) ~4'4~ 9 CMV (archival paraffin-embedded tissues)z4 9 HHV-6, HHV-8/KSHV (Kaposi's sarcoma, lymphomas, etc.) z~ 9 HSV-DNA/RNA (trigeminal ganglia, disseminated encephalitis) 26,34,35 9 LGV (lymphogranuloma venerum) (Bagasra et al.) 51 Oncogene/tumour suppressor genes 9 p53 mutations (paraffin-embedded tissue) (Bagasra et al.) 5~ 9 Gene re-arrangements (t11:22 in PNET, t14:18 in follicular lymphoma)41 9 ras mutations (H, Ki, N-ras, codon 12, 13 and 61) 9 Chromosome mapping (PRINS and cycling PRINS) 33 9 T-cell receptor gene re-arrangements and immunoglobulin heavy and light V chains3~ Growth factors, markers of malignancy and other biological markers 9 Metalloproteinases and their inhibitors ]7 9 EGF receptor mRNA expression3s 9 Endothelial receptor mRNA 29 9 Nitric oxide synthase in multiple sclerosis5
Table 2--Potential uses of in situ amplification techniques in pathology Detection of viral genes in tissues, e.g. HPV, EBV, CMV, KSHV, HIV 1 and 2 Detection of bacterial species, e.g. Mycobacterium tuberculosis, M. leprae, M. bovis, etc. Detection of mammalian structural genes in embryogenesis and dysmorphogcnesis Detection of point mutations in oncogenes and turnout suppressor genes, e.g. ras, p53 Single cell allelic discriminatkm and individual cell haplotyping, e.g. cystic fibrosis, thalassaemias, etc. Detectkm of chromosomal translocations, e.g. t l 1:22, t14:18, etc. Quantitative in situ amplification for viral load and oncogene expression (IS-TaqMAN PCR) Assessment of loss of heterozygosity (LOH) and allelic imbahmce Forensic identification of tissues, e.g. HLA DQ, etc
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