DNA Probes in Diagnostic Pathology

HUBERT J. WOLFE, M.D.

Molecular pathology has become firmly established as a dis- Pathology Department, New England Medical Center
tinctive discipline in medicine. It has introduced radical Hospitals, Boston, Massachusetts
changes in concepts of disease causation and in classification of
disease states affecting humans and other organisms. In addi-
tion, molecular pathology represents a "new" diagnostic tech-
nology with many potentials that have been heretofore un- Methods
tapped. This overview provides a discussion of the use of DNA
probes in the study of human diseases. The role of detectable A variety of molecular probes are available (Table 1).
genetic abnormalities in pathogenesis will be considered, as In all instances, the strength of hybridization between
well as their possible impact on nosology and disease classifi- the nucleic acid sequences and probes is proportional to
cation. (Key words: In situ hybridization; DNA and RNA
probes; Nucleic acids) Am J Clin Pathol 1988;90:340-344 their degree of homology. Stringent conditions of hy-
bridization are set so as to ensure a high degree of speci-
Author Biography: Hubert J. Wolfe, M.D. ficity for the stable complexes formed. Among the cate-
gories of probes, double-stranded DNA probes are the
Dr. Wolfe graduated from the University of Louisville most commonly used. By comparison, single-stranded
School of Medicine and did his internship at the Boston cRNA probes are more sensitive but require RNase-free
City Hospital and his residency in Pathology at the conditions for their preparation. Synthetic oligonucleo-
Massachusetts General Hospital, where he was a Fellow tides may offer the ideal probes for the future because
of the Medical Foundation. He is currently Professor of they are synthesized de novo and therefore have an ab-
Pathology and Associate-Pathologist-in-Chief at Tufts- solute definition of nucleotide sequence, a high degree of
New England Medical Center. His research interests are consistency in production, and a total absence of vector
primarily in the field of endocrine pathology and pren- sequences. With all probes, sensitive labeling techniques
eoplasia and early neoplasia. Dr. Wolfe is a member of have been developed, usually with direct incorporation
the Editoral Board of Virchows Archiv. B. Cell Pa- of the label into the probe (Table 1). Currently, the most
thology. commonly used and most sensitive labels are radioisoto-
pic, but nonisotopic markers are being developed that
will soon match or exceed the isotopic methods for sen-
WE HAVE WITNESSED dramatic developments in sitivity and offer greater stability and ease of handling
the discipline of molecular biology. These advances and more rapid signal detection.
have revolutionized the field of genetics and placed the Molecular probes may be used effectively with a wide
human genome squarely at the center of molecular bio- range of methods, each exploiting the strategy of molec-
logic research.' The new concepts and molecular tools ular hybridization in a slightly different way.7 Four
have attracted increasing interest from pathologists, and commonly used methods are filter hybridization,
recent biotechnical advances have now firmly estab- Southern blot, Northern blot, and in situ hybridization.
lished molecular pathology as both a basic science and Filter hybridization is the simpliest of these techniques
diagnostic clinical entity. The speed of these advances and involves the placement of denatured DNA or RNA,
has exceeded expectations partly because of rapid pro- extracted from cells or tissues, on a membrane that acts
gress in recombinant DNA technology. Molecular as an inert support for the nucleic acid and makes it
probes are key tools of this technology. They have well- easily accessible for hybridization with the labeled
defined physical and chemical properties that govern the probes. The slot blot is the most effective modification
basic principles by which molecular hybridization of this technique, allowing for the rapid processing on a
occurs and make such probes sensitive and highly spe- single membrane of more than 70 different samples for
cific biologic markers of gene expression. detection and quantitation of nucleic acids.
Another more elaborate but very important method is
the Southern blot. High molecular weight DNA is di-
gested with restriction endonuclease enzymes that clip
. Address reprint requests to Dr. Wolfe: Pathology Department, New
England Medical Center Hospitals, 750 Washington Street, Boston, the DNA into different-sized pieces. The resultant re-
Massachusetts 02111. striction fragments are separated by gel electrophoresis

340
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 Vol. 90 • No. 3 PATHOLOGY PATTERNS 341
Table 1. Molecular Probes and Labeling Techniques In many ways the most novel and potentially power-
ful of these methods is in situ hybridization. This tech-
I. Probes
a. Double-stranded DNA
nique is derived from the Southern and Northern blot
b. Synthetic oligonucleotides technology but offers the unique advantage of detecting
c. Single-stranded cRNA specific genomic DNA or specific messenger RNA se-
quences in intact tissue sections, dispersed cells, or chro-
II. Labeling techniques
a. Isotopic mosomal preparations (Fig. 2). In situ hybridization
32 33 3
P, S, H, l4C, l25I weds molecular biology and morphology into a single
b. Nonisotopic discipline and offers the pathologist the opportunity to
Biotin, fluorochromes, enzymes, and antibodies
study patterns of gene expression in heterogenous cell
populations. In addition, although the conventional
Southern or Northern blot analysis may require 10-20
according to size, with the longer fragments moving million cells to generate an adequate signal, in situ hy-
more slowly than the shorter fragments, thereby segre- bridization can detect hybridization signal at the level of
gating the DNA into a series of bands (Fig. 1). The re- the single cell in tissue sections and can detect signal at
sultant bands are transferred onto an inert membrane the level of single gene copy in chromosomal prepara-
for hybridization reaction with a molecular probe in a tions. The implications are enormous, and the potential
manner similar to that of the less sensitive but simplier applications to pathobiology are nearly limitless! The
filter hybridization technique. New methods have been sensitivity of in situ hybridization is limited by several
developed to extend the sensitivity of Southern blot for variables, including preservation of nucleic acid during
the detection of smaller quantities of genomic DNA. fixation, accessibility of molecular probe to the nucleic
The technique of in vitro DNA amplification with the acids during hybridization, specific activity of labeled
use of the polymerase chain reaction has proved particu- probe, and the sensitivity of the detection system. Both
larly valuable in this regard. The method offers the op- isotopic and nonisotopic detection systems have been
portunity to detect a much smaller number of copies of used successfully with all types of molecular probes for
a specific genomic DNA sequence by undertaking in in situ hybridization. Radioisotopically labeled cRNA
vitro replication to amplify the sequence one million- probes currently appear to be the most sensitive, for the
fold before the Southern blot analysis. The amplification detection of low copy number (< 100) of specific mRNA
technique is based upon the use of two or more primers and DNA sequences in tissue sections. Single-stranded
flanking both ends of the selected fragment of genomic cRNA probes have several important characteristics, in-
DNA and then copying the DNA template with high cluding high specific activities, greater hybridization ef-
fidelity by use of the polymerase chain reaction in re- ficiency, and the opportunity to greatly reduce back-
peated amplification cycles.8 Such a method allows for ground noise by removal of all single-stranded nonhy-
the detection of mutant sequences of single copy genes bridized radiolabeled cRNA probes from tissues by
in a few hundred cells. Northern blot analysis is a very ribonuclease digestion.4 Different conditions may call
similar to the Southern blot, but the technique is tai- for different combinations of probes and labels. For the
lored for detection and quantitation of messenger RNAs use of in situ hybridization with flow cytometry, fluoro-
rather than genomic DNA. chrome labeled DNA probes have proved to be the most

FlG. 1. In Southern blot analysis, restriction

endonucleases are first used to cut the DNA
into multiple subgenomic fragments of varying
size. The DNA digest is then electrophoresed,
transferred to a membrane, and hybridized with
labeled probes. Signal detection creates bands
representing specific genomic DNA sequences
complementary to the labeled probe.
High Molecular DNA Fragments Transfer and
Weight DNA for Electrophoresis Detection of
+ Hybridization
Endonuclease Signal for
Digestion Specific DNA

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 342 WOLFE AJ.C.P. • September 1988

successful with intact cells and offer great potential for cRNA cDNA
chromosomal preparations.2 The potential of flow cy-
tometry with in situ hybridization at the cellular and
chromosomal level is extraordinary and represents one
of the new rapidly developing technologies. In situ hy-
bridization studies require stringent positive and nega-
tive controls even with well-characterized molecular
probes. Positive controls include Northern and/or
Southern blot analysis of the same tissue and in most
instances immunocytochemical detection of gene prod-
uct coded for by the specific mRNA in positively hybrid-
ized cells. Negative controls include use of probes to
genes known to not be expressed in the cells and tissues
under study.

Infectious Diseases
Molecular probes are ideally suited for the study of a
wide range of infectious agents. These markers are rap-
idly becoming the gold standard for their detection and
classification and may be used in the future to similarly
determine sensitivity to antimicrobial agents. This tech- FIG. 2. A schematic representation of in situ hybridization. Asterisks
nology is particularly valuable in viral infections, in identify isotopic or nonisotopic probe labels used to detect and visual-
ize hybridization sites within the cell.
which probes have the ability to rapidly detect both ac-
tive and latent infections. Such probes have great poten-
tial to provide insight into the molecular mechanisms of
viral-induced cell injury and to determine the role of A less dramatic but similar situation exists in the gen-
viruses in developmental abnormalities, chronic degen- ital tract, where at least 14 different HPV strains have
erative diseases, and oncogenesis. The probes used may been detected but only a few seem oncogenic. Strains 16,
represent the entire viral genome or sequences homolo- 18, 33, and 35 are associated with cervical dysplasia, and
gous to only a portion of the viral genome, i.e., subge- 16, 18, and 33 have been detected in a high proportion
nomic probes. of invasive squamous cell carcinomas of the cervix. In
situ hybridization offers a unique opportunity to apply
Subgenomic probes have been particularly valuable strain-specific probes to exfoliative cervical cytologic
for strain-specific detection of viruses. Their use can studies and cervical biopsies for the simultaneous detec-
make Southern blot analysis for strain-specific identifi- tion of dysplasia and high-risk strain-specific infections.
cation far easier to interpret. In addition, these probes
may be used effectively for detection and typing by in
situ hybridization. Strain-specific detection is important Genetic Disorders
for both clinical and epidemiologic studies. Human pa- Although conventional cytogenetic techniques permit
pilloma virus (HPV) illustrates some of the advantages.5 the recognition of chromosomal deletions, transloca-
HPV is a heterogenous group of DNA viruses with a tions, or duplications, molecular biologic techniques
circular double-stranded genome well known to infect have contributed to the tools necessary for the detection
squamous epithelium and produce a wide range of pro- of much smaller but equally important genetic errors,
liferative lesions. Currently, more than 50 different even at the level of a single base pair alteration in DNA.
strains of HPV are recognized, each of which has had its Specially constructed oligonucleotide probes can now
DNA sequences cloned. Infection by some strains is directly target such point mutations on Southern blot
clearly associated with an increased frequency of malig- analysis of genomic DNA from affected persons. In situ
nant transformation in the infected epithelial cells. Pos- hybridization, which has emerged as the most powerful
sibly the most dramatic example is noted with epider- tool for chromosomal mapping of single and multiple
modysplasia verruciformis, where infection with HPV copy genes in the haploid genome, has also proved to be
types 5, 8, and 14 is associated with the development of of great value to the cytogeneticist for the delineation
squamous cell carcinoma of the skin in a high fre- and classification of many genetic diseases. The value of
quency. these techniques for prenatal and postnatal detection of

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an ever-increasing number of genetic diseases and their this molecular arena further underscores the intricate
carrier states cannot be overestimated. Indeed, these and complex nature of neoplasia. The pathologist has
techniques are currently being used for genetic counsel- much to contribute to the unraveling of the role of on-
ing in a growing number of instances.6 The stage is now cogenes by examining their expression during the course
set for a clearer understanding of the molecular mecha- of stepwise morphologic events that occur in neoplastic
nisms of genetic diseases, including analysis of factors progression. Such problems are exquisitely tailored to
that, from person to person, may modify clinical fea- the use of molecular probes and especially to in situ
tures, age of onset, etc. hybridization.
Much less is known about the molecular genetics of In selected instances, overexpression of certain onco-
common diseases that are thought to not be caused by a genes has been correlated with biologic behavior of the
single gene defect but result from the interplay between neoplasm. Salmon and associates first reported a corre-
multiple genes and environmental factors, i.e., multi- lation between overexpression of the oncogene, neu, in
factoral or polygenic diseases. Coronary heart disease, breast carcinoma and time to relapse.10 In their study of
diabetes mellitus, essential hypertension, schizophrenia, human primary breast carcinomas, they found a 2- to
and senile dementia are examples of common diseases 20-fold overexpression of neu oncogene in one-third of
of the adult with frequent family clustering but are not all of their patients. Statistical analysis demonstrated
inherited as single-gene, monogenetic disorders.9 that the increased number of copies of neu gene and its
Clearly, new approaches are needed to define this com- corresponding mRNA per tumor cell is a prognostic
plex interplay between gene expression and environ- indicator equal to that of pathologic staging and hor-
mental factors at the molecular level. Such studies could monal receptor status. Other workers have now inde-
lead to predictive screening for early detection of people pendently confirmed neu overexpression as an indicator
at high risk and offer the opportunity to modify environ- of overall survival and time to recurrence. Similar re-
mental factors and thereby alter disease course in condi- sults have been reported in neuroblastoma with the on-
tions that affect substantial segments of the population. cogene N-myc, where overexpression correlated with
advanced-stage disease, suggesting it to be an indicator
Neoplasia of poor prognosis.3 In a similar manner, Minna and
Southern and Northern blot analysis of tumor tissues Johnson have described a correlation between a C-myc
and chromosomal mapping by in situ hybridization oncogene overexpression and poor prognosis in ad-
have been invaluable in characterizing specific translo- vanced small cell carcinoma of the lung. Although there
cations, amplifications, deletions, and point mutations has not been a systematic study and correlation of on-
that have been identified as key events to the develop- cogene overexpression and/or structural mutation with
ment of neoplasia. In many instances these genetic ab- clinical course in a wide range of tumors, it is clear that
normalities are typical or pathogenomic of a particular this strategy has already proved beneficial to molecular
neoplasm or tumor-associated syndrome. Such analyses biologists, pathologists, and clinicians alike, and it is
will play an increasingly important role in the diagnosis very likely that other valuable clinical correlations and
and classification of epithelial and nonepithelial neo- new insights into the biologic roles of oncogenes will
plasms. follow from additional studies. Possibly one of the most
Currently, a small subset of cellular genes associated exciting potentials for diagnostic pathologists is the ap-
with cellular proliferation and differentiation, i.e., pro- plication of in situ hybridization for detection of onco-
tooncogenes, are the focus of intense investigation in gene expression in preneoplasia and early neoplasia.
oncogenesis. Basic research strongly suggests that pro- This techniqe, carefully controlled with correlative
tooncogenes are frequent targets for various genetic al- Southern and Northern blot analysis, may offer an op-
terations that are key events in the course of neoplastic portunity to establish new criteria for the detection of
progression." These alterations or activations can be borderline morphologic lesions that currently remain a
caused by structural changes in the genomic DNA, dilemma for the pathologist and clinician.
which may result in point mutations or overexpression. Molecular probes can also be extremely valuable in
These changes may result in the transformation of cells the assessment of the degree and direction of differen-
into their neoplasic counterparts. Although an ever-in- tiation in various neoplasms of epithelial and nonepi-
creasing number of protooncogenes have been identi- thelial origin. For example, Berge-LeFranc and co-
fied, their normal biologic functions and their roles in workers demonstrated that the well-differentiated papil-
the development of neoplasia are poorly understood. In lary and follicular neoplasms of the thyroid express very
addition, with the emergence of the concept of antion- high copy level of tissue-specific thyroglobulin mRNA,
cogenes, the biologic significance of their interaction in whereas those of poorly differentiated carcinomas ex-

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 WOLFE A.J.C.P. • September 1988
344
press dramatically lesser amounts of thyroglobulin-spe- 3. Brodeur GM, Seeger RC, Sather H, et al. Clinical implications of
oncogene activation in human neuroblastoma. Cancer
cific mRNA, thus, adding a new and objective parame- 1986;58:541-545.
ter to the subjective criteria of histologic grading of neo- 4. Hoefler H, Childers H, Montminy MR, Lechan RM, Goodman
plasms. RH, Wolfe HJ: In situ hybridization methods for the detection
of somatostatin mRNA in tissue sections using anti-sense
mRNA probes. Histochem J 1986;18:597-604.
Summary 5. Lutzner MA: Papilloma virus and neoplasia in man. In: Fenogho-
Molecular pathology is now firmly established as a Drush CM, Weinstein RS, Kaufman N, eds. New concepts in
neoplasia as applied to diagnostic pathology. Baltimore: Wil-
discipline in medicine. It is creating radical changes in liams and Wilkins, 1986:126-170.
our concepts of pathogenesis and our classification of 6. Martin JB: Molecular genetics: Application to the clinical neuro-
diseases as well as a whole new diagnostic technology. As sciences. Science 1987;238:765-771.
7. Maniatis T, Fritsch E, Sambrook J: Molecular cloning: A labora-
the structure and location of new genes are discovered, tory manual. Cold Spring Harbor, NY: Cold Spring Harbor
the pathologist will be in a pivotal position between the Laboratory, 1982.
molecular biologists and clinicians to help unravel the 8. Saiki RK, Scharf S, Falooma F, et al: Enzymatic amplification of
beta-globulin genomic sequences and restriction site analysis
function of these genes in health and disease and to for diagnosis of sickle cell anemia. Science 1985;230:1350-
apply the findings directly to the practice of pathology. 1354.
9. Scott J: Molecular genetics of common diseases. Br Med J
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