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Rallying call or death knell?
Human PATHOLOGY VOLUME 25
June 1994
NUMBER 6
Editorial Rallying Call or Death Knell? It is not often that an individual is given the op portunity of hearing the bell tolling his or her prospective demise. However, for some little while, such a bell has not only been heralding a revolution in the world of medicine and ushering in new concepts and technology but also sounding a warning for anatomical pathology with an intensity that we ignore at our collective peril. This issue of HUMANPATHOLOGYfeatures a symposium of articles outlining many of the fundamental (and already familiar) molecular biological techniques that are now commonly used in the routine analysis of human pathological material. These articles show the importance and power of the new analytical techniques. Recent technological developments yield rapid generation of results with relative simplicity of test performance and thus present opportunities to make rapid and hmdamental advances toward understanding the path@ logical basis of many human diseases as well as to contribute to biologically appropriate management. With respect to the current practice of pathology, molecular biological techniques have been viewed as a doubleedged sword. For these new opportunities to be realized molecular biological techniques must be incorporated into the practice of pathology. Otherwise, our role as tissue diagnosticians will be jeopardized and the relationship between pathologists and clinicians will be altered irrevocably. Cancer is an obvious field in which the identification of genetic changes is about to have a major impact on diagnostic pathology. Distinction of benign tissue from malignant or premalignant lesions, rapid and accurate screening of individuals to detect their predisposition to particular malignancies, and the prediction of tumor behavior and prognosis are three areas in which molecular technology has begun to alter diagnosis and management. Our present knowledge of the natural history of the accumulation of mutations during tumor progression is restricted, and the analysis of timing, sequence, and disease specificity of mutations will clearly augment that knowledge. It is apparent that some mutations arise early during tumor progression, whereas others accumulate at later stages. Clinical fol-
low-up, in conjunction with pathological diagnosis and mutational analysis, will be required to elucidate the natural history of individual lesions and their particular genetic changes. Analysis of specific mutations is likely to be a powerful approach to screening for specific cancers. Within the near future we shall know which combinations of mutations reliably distinguish between benign and malignant lesions. In tissues such as the colon, breast, and prostate, molecular analysis will be used routinely to confirm the diagnosis. For example, the detection of Krus mutations, either alone or in combination with other molecular changes, might become an important screening technique for colonic cancer or a prognostic indicator in pancreatic adenocarcinoma. More than 90% of pancreatic ductal carcinomas contain specific mutations that accurately differentiate these tumors from other types of periampullary cancers. The prognostic power of specific mutations is exemplified by KTUSmutations in adenocarcinomas of the lung where their presence is strongly predictive of a markedly worse prognosis. The technique known as single-strand conformation polymorphism is a powerful method of identifying point mutations in pathological tissues but does not yield their sequences. To obtain a precise sequence either conventional sequencing or screening with a series of oligonucleotides containing that mutation is required. For the pathologist the particular strength of the polymerase chain reaction is the requirement for only minute quantities of tissues comprising only a few cells, such as may be obtained by fine-needle aspiration under radiographic control. Polymerase chain reaction products are detectable by a variety of methods, including a simple dot-blot analysis or separation of amplified products and visualization using polyacrylamide gel electrophoresis. Hybridization with oligonucleotide probes labeled with radioactive, fluorescent, or enzymatic tags may be used to increase the sensitivity of individual assays and to confirm the specificity of the amplified product. In addition to applications in cancer pathology, these molecular techniques provide rapid
553
HUMAN
PATHOLOGY
Volume 25, No. 6 (June 1994)
methods for early recognition of graft-versus-host disease and for distinguishing among inflammatory lesions, particularly those of viral origin. Specific strains of viruses may be typed on the same extracted genetic material and at the same time. In situ hybridization is a remarkably powerful tool for assessing gene expression at the cellular level in intact tissue slices. In contrast to immunohistochemistry, which identifies the translated protein product of cellular genes, DNA in situ hybridization identifies genomic mutations and quantifies alterations of gene or chromosome copy number. Clonality of cells and heterogeneity of tissues can be identified. Hybridization for specifically transcribed messenger RNAs (mRNAs) extends conventional molecular techniques, such as Northern blot analysis, in a manner analogous to the way in which immunohistochemistry extends Western blot assays. Both immunohistochemistry and DNA or RNA in situ hybridization provide data on individual cells rather than on an average of the total cellular population. In addition, immunohistochemistry and RNA in situ hybridization are complementary: changes in mRNA levels determined by Northern blot and quantitative in situ hybridization assays more closely correlate with functional activity than do the results of immunohistochemistry for the demonstration of translational products. High levels of a particular protein, determined by immunohistochemistry, could represent increased synthesis, accumulation of material produced elsewhere, or decreased degradation of that protein, whereas mRNA levels indicate gene transcriptional activity. In situ hybridization provides a critical method for the analysis of heterogeneity in tumors containing cells at different phases of neoplastic progression, at multiple stages of differentiation, and at various levels of functional activity. The impressive power of molecular biology is that it is capable of revealing previously hidden predispositions to many diseases that have hitherto eluded comprehension, and not just cancer or the so-called “inherited disorders.” Much of the new molecular biological information that is currently transforming the practice of medicine, including anatomical pathology, is being collected with unforeseen rapidity. It is likely that the Human Genome Project, in which the location, structure, and (eventually) function of every
554
human gene is being mapped, will be completed several years ahead of schedule. The data obtained from this project will then allow identification and rapid correlation between a wide range of human diseases and the regions of the human genome important to those conditions. Unfortunately, much of the new information about human diseases is not coming from anatomical pathologists but from research laboratories organized by clinicians. If current trends in the application of molecular genetic techniques to pathological material continue, the anatomical pathologist will no longer be required to make many of the fundamental diagnoses and much of the input from morphological evaluation will be lost. Instead, the primary diagnosis of conditions as varied as hepatitis C, chronic pancreatitis, and prostate cancer will be made by the clinicians, reducing the role of the pathologist to that of an assessment of the extent of tissue involvement by a particular disease or its tissue distribution. Pathologists have already allowed the initiative for much of the new molecular enterprise to pass from their hands into what might be viewed as the “public medical” domain. If we fail to seize the opportunity presented, we will lose our central role as the translators of our existing expertise in tissue morphology and pathogenesis into the new world of molecular diagnosis. That expertise is essential for accurate localization and interpretation of the results of the new methods. To survive in this brave new world of molecular medicine anatomical pathologists must regain the initiative as laboratorybased investigators of the pathological basis of disease. Ask not for thee!
forwhom
the bell tolls-it
might be tolling
CHRISTOPHERS. FOSTER, MD, PHD, MRCPATH Royal Postgraduate Medical School Hammersmith Hospital London, UK
MILTONJ. FINEGOLD, MD Baylor College of Medicine Texas Children’s Hospital Houston, TX
SANDRAR. WOLMAN,MD Wayne State University School of Medicine Detroit, MI
June 1994
NUMBER 6
Editorial Rallying Call or Death Knell? It is not often that an individual is given the op portunity of hearing the bell tolling his or her prospective demise. However, for some little while, such a bell has not only been heralding a revolution in the world of medicine and ushering in new concepts and technology but also sounding a warning for anatomical pathology with an intensity that we ignore at our collective peril. This issue of HUMANPATHOLOGYfeatures a symposium of articles outlining many of the fundamental (and already familiar) molecular biological techniques that are now commonly used in the routine analysis of human pathological material. These articles show the importance and power of the new analytical techniques. Recent technological developments yield rapid generation of results with relative simplicity of test performance and thus present opportunities to make rapid and hmdamental advances toward understanding the path@ logical basis of many human diseases as well as to contribute to biologically appropriate management. With respect to the current practice of pathology, molecular biological techniques have been viewed as a doubleedged sword. For these new opportunities to be realized molecular biological techniques must be incorporated into the practice of pathology. Otherwise, our role as tissue diagnosticians will be jeopardized and the relationship between pathologists and clinicians will be altered irrevocably. Cancer is an obvious field in which the identification of genetic changes is about to have a major impact on diagnostic pathology. Distinction of benign tissue from malignant or premalignant lesions, rapid and accurate screening of individuals to detect their predisposition to particular malignancies, and the prediction of tumor behavior and prognosis are three areas in which molecular technology has begun to alter diagnosis and management. Our present knowledge of the natural history of the accumulation of mutations during tumor progression is restricted, and the analysis of timing, sequence, and disease specificity of mutations will clearly augment that knowledge. It is apparent that some mutations arise early during tumor progression, whereas others accumulate at later stages. Clinical fol-
low-up, in conjunction with pathological diagnosis and mutational analysis, will be required to elucidate the natural history of individual lesions and their particular genetic changes. Analysis of specific mutations is likely to be a powerful approach to screening for specific cancers. Within the near future we shall know which combinations of mutations reliably distinguish between benign and malignant lesions. In tissues such as the colon, breast, and prostate, molecular analysis will be used routinely to confirm the diagnosis. For example, the detection of Krus mutations, either alone or in combination with other molecular changes, might become an important screening technique for colonic cancer or a prognostic indicator in pancreatic adenocarcinoma. More than 90% of pancreatic ductal carcinomas contain specific mutations that accurately differentiate these tumors from other types of periampullary cancers. The prognostic power of specific mutations is exemplified by KTUSmutations in adenocarcinomas of the lung where their presence is strongly predictive of a markedly worse prognosis. The technique known as single-strand conformation polymorphism is a powerful method of identifying point mutations in pathological tissues but does not yield their sequences. To obtain a precise sequence either conventional sequencing or screening with a series of oligonucleotides containing that mutation is required. For the pathologist the particular strength of the polymerase chain reaction is the requirement for only minute quantities of tissues comprising only a few cells, such as may be obtained by fine-needle aspiration under radiographic control. Polymerase chain reaction products are detectable by a variety of methods, including a simple dot-blot analysis or separation of amplified products and visualization using polyacrylamide gel electrophoresis. Hybridization with oligonucleotide probes labeled with radioactive, fluorescent, or enzymatic tags may be used to increase the sensitivity of individual assays and to confirm the specificity of the amplified product. In addition to applications in cancer pathology, these molecular techniques provide rapid
553
HUMAN
PATHOLOGY
Volume 25, No. 6 (June 1994)
methods for early recognition of graft-versus-host disease and for distinguishing among inflammatory lesions, particularly those of viral origin. Specific strains of viruses may be typed on the same extracted genetic material and at the same time. In situ hybridization is a remarkably powerful tool for assessing gene expression at the cellular level in intact tissue slices. In contrast to immunohistochemistry, which identifies the translated protein product of cellular genes, DNA in situ hybridization identifies genomic mutations and quantifies alterations of gene or chromosome copy number. Clonality of cells and heterogeneity of tissues can be identified. Hybridization for specifically transcribed messenger RNAs (mRNAs) extends conventional molecular techniques, such as Northern blot analysis, in a manner analogous to the way in which immunohistochemistry extends Western blot assays. Both immunohistochemistry and DNA or RNA in situ hybridization provide data on individual cells rather than on an average of the total cellular population. In addition, immunohistochemistry and RNA in situ hybridization are complementary: changes in mRNA levels determined by Northern blot and quantitative in situ hybridization assays more closely correlate with functional activity than do the results of immunohistochemistry for the demonstration of translational products. High levels of a particular protein, determined by immunohistochemistry, could represent increased synthesis, accumulation of material produced elsewhere, or decreased degradation of that protein, whereas mRNA levels indicate gene transcriptional activity. In situ hybridization provides a critical method for the analysis of heterogeneity in tumors containing cells at different phases of neoplastic progression, at multiple stages of differentiation, and at various levels of functional activity. The impressive power of molecular biology is that it is capable of revealing previously hidden predispositions to many diseases that have hitherto eluded comprehension, and not just cancer or the so-called “inherited disorders.” Much of the new molecular biological information that is currently transforming the practice of medicine, including anatomical pathology, is being collected with unforeseen rapidity. It is likely that the Human Genome Project, in which the location, structure, and (eventually) function of every
554
human gene is being mapped, will be completed several years ahead of schedule. The data obtained from this project will then allow identification and rapid correlation between a wide range of human diseases and the regions of the human genome important to those conditions. Unfortunately, much of the new information about human diseases is not coming from anatomical pathologists but from research laboratories organized by clinicians. If current trends in the application of molecular genetic techniques to pathological material continue, the anatomical pathologist will no longer be required to make many of the fundamental diagnoses and much of the input from morphological evaluation will be lost. Instead, the primary diagnosis of conditions as varied as hepatitis C, chronic pancreatitis, and prostate cancer will be made by the clinicians, reducing the role of the pathologist to that of an assessment of the extent of tissue involvement by a particular disease or its tissue distribution. Pathologists have already allowed the initiative for much of the new molecular enterprise to pass from their hands into what might be viewed as the “public medical” domain. If we fail to seize the opportunity presented, we will lose our central role as the translators of our existing expertise in tissue morphology and pathogenesis into the new world of molecular diagnosis. That expertise is essential for accurate localization and interpretation of the results of the new methods. To survive in this brave new world of molecular medicine anatomical pathologists must regain the initiative as laboratorybased investigators of the pathological basis of disease. Ask not for thee!
forwhom
the bell tolls-it
might be tolling
CHRISTOPHERS. FOSTER, MD, PHD, MRCPATH Royal Postgraduate Medical School Hammersmith Hospital London, UK
MILTONJ. FINEGOLD, MD Baylor College of Medicine Texas Children’s Hospital Houston, TX
SANDRAR. WOLMAN,MD Wayne State University School of Medicine Detroit, MI