Application of DNA Probes to Hematology: an Overview with Selected Examples* GEORGE H

Application of DNA Probes to Hematology: An Overview with Selected Examples* GEORGE H. BARROWS, M.D.f

Department o f Pathology, Saint Francis Medical Center, Hartford, CT 06105 and Departments o f Pathology and Laboratory Medicine, University o f Connecticut School o f Medicine, Farmington, CT 06032

ABSTRACT Recent advances in molecular genetics have allowed development of short segments of desoxyribonucleic acid (DNA) which are complemen tary to active genetic sites. By pairing normal DNA with these “molecular probes”, the presence of specific genetic sequences may be detected in patient samples. The majority of hematologic diseases arise from heredi tary errors, neoplastic change or parasitic organisms. These types of dis orders are particularly suited to diagnosis and management using detec tion of abnormal DNA sequences. Sensitive laboratory techniques employing these molecular probes are now available directly to detect tar get DNA sequences, assess abnormal short sequences of DNA, and evalu ate the presence of abnormal DNA from its transcription products. Using DNA polymerase amplification, the sensitivity of these methods approaches its theoretical limitation of a single gene pair. In hematology, a prevalent application has been the detection of clonal molecular rear rangements which serves as a marker for lymphocytic neoplasia. Other applications of these techniques include the detection of abnormal chro mosomal regions such as the Philadelphia chromosome and the detection of deletions of DNA which occur in such diseases as the myeloid dysplas- tic syndrome. As more human genetic sequences are known, it seems likely that these techniques will become prevalent among methods of lab oratory testing.

* Presented at the meeting of the Association of Introduction Clinical Scientists, Hartford, CT, November 1988. t To whom reprint requests should be addressed The development of DNA probes at the Department of Pathology and Laboratory promises to provide a significant advance Medicine, Saint Francis Medical Center, 114 Wood land Street, Hartford, CT 06105. in many areas of hematology. In this Please see glossary of terms on page 145. review, a brief history of these tech-

139 0091-7370/89/0500-0139 $01.20 © Institute for Clinical Science, Inc. 140 BARROWS niques will be presented and several To be active, it was found that double specific examples will be used to demon stranded DNA needed to be separated strate current applications. into single strand to allow exposure of a A full century of experimental work single genetic sequence. A number of preceded the development of specific synthetic methods to allow duplication genetic probes .8 Although DNA was and synthesis of DNA fragments were known to be a major constituent of cell developed based on enzymatic systems nuclei since 1869, its role as the media found in bacteria. While most genetic tor of genetic change was first elucidated activity involved DNA to DNA and DNA in 1944 by Dr. Owald Avery and asso to ribonucleic acid (RNA) transcription, ciates. During the 1950s, the molecular there were viral systems which actively basis of inheritance was clarified by performed DNA synthesis from RNA description of DNA base pair structure. base pairs using a “reverse transcriptase” After description of the double helix enzyme. Using these methods in vitro, structure of the DNA molecule was com direct manipulation of genetic material pleted by Watson and Crick in 1953, the became possible. first studies utilizing synthetic manipula At this point, there was a hiatus tions of genetic material became possi because no facile methods were available ble. to decode the simple complexity of the During the 1960s the biochemical DNA genetic sequence which consisted events in transcription of the genetic of four nearly identical base pairs. Con code into somatic expression were eluci sidering that a DNA molecule is over 6 dated: (1) The transcription of areas of feet long with six billion base pairs of DNA to mRNA; (2) M igration of th e four monomeric units, it is not surprising mRNA to the cytoplasmic ribosomes; that chemical attempts at sequencing and (3) Incorporation of genetic informa were not immediately successful. The tion from mRNA into proteins by tRNA discovery of specific “restriction endonu “codons” into amino acid sequences. cleases” which allow cleavage of molecu The identification of chromosomes as lar DNA at selected sites led to the next the carriers of genetic material marked series of advances in molecular genetics. the beginning of Molecular Genetics as a Over 200 of these enzymes are now separate field of the medical sciences. It known, each of which is active at a spe was not until 1956 that the number of cific DNA sequence. The enzymes are human chromosomes was firmly estab named using a short hand denoting the lished at 46. Using only photographic genus and species of bacteria from which techniques, chromosomal alterations they were isolated and a roman numeral were detected which could be correlated suffix describing the type of endonu with genetic defects such as mongolism clease activity which they exhibit. The (trisomy 2 1 ) and diseases such as chronic use of these enzymes to digest homoge myelocytic leukemia (9;22 translocation- neous mixtures of DNA allows native “Philadelphia Chromosome”). With DNA to be separated into smaller frag improvements in chromosomal mapping ments. These fragments may then be using high resolution banding tech separated by molecular weight using niques, it became possible to detect agarose electrophoresis calibrated by abnormal chromosomes with increasing synthetic DNA markers of known size. sensitivity. In diseases such as non- Given the enormous number of frag Hodgkins lymphoma, for example, over ments possible from any given digest (on 95 percent of cases have had structural the order of 1 0 million), this still pro alterations identified. vides little hope of identification of single APPLICATIONS OF DNA PROBES IN HEMATOLOGY 141 base pair alterations. However, once a cells which express DNA complemen gene segment sequence is known, small tary to the DNA probe are specifically segments of DNA between 20 and 1000 labeled. This allows identification of cells base pairs may be manufactured which which have the target DNA sequences allow pairing to known segments of expressed and morphologic evaluation of genetic material. These small probes will their location provides insight regarding specifically pair only with complemen processes which may be present. tary sites. Since a great deal of signal The most extensive application to date amplification is necessary, most probes has been the utilization of a combination have been labelled with very active of restriction endonuclease digestion and radioisotopes (32P and a5S) or biotin/avi- DNA probe localization utilizing the din complexing methods to allow detec technique developed by E. M. South tion. The radioisotopes have an advan ern, of Dallas, Texas. The DNA is iso tage of facile application to probes and lated from cell lysates, denatured into great sensitivity, but have a very short single strands, and digested with a spe shelf life and the attendant problems cific restriction endonuclease. The frag associated with handling radioactive ments of DNA which are produced with material. Increasing availability of bio- this procedure are separated in bands by tin/avidin labeled probes of satisfactory agarose electrophoresis on the basis of sensitivity promises to replace radioiso molecular size. The bands are then topic methods for most applications. transferred by blotting the agarose jell to a nylon or nitrocellulose membrane Review of Methods which will support hybridization of the DNA fragments with specific DNA com Several procedures allow these plementary probes. After the probes genetic probes to be used in diagnostic have been hybridized to target techniques. The probes may be hybrid sequences, the bands having localization ized to DNA lysed from cells. The DNA are developed using autoradiography or from the cells under study which have immunochemistry. By choosing endonu hybridized strands of the probe then cleases which are active near a target site are localized on filter paper. The pres of interest, characteristic alterations in ence of this complexed DNA is detected the migration of the target DNA seg by autoradiography or immunochemical ments occur. Since the probes will only detection using biotin/avidin complexing react with the areas of interest, specific methods. This “Dot-Blot” technique variations in DNA may be characterized. allows identification of these specific This method has proven to be very marked gene sites. Uses include identifi reproducible and promises extension to cation of viruses, parasites, abnormal many aspects of medical science. DNA of genetic diseases or altered Similar blot transfers and probe label somatic DNA. ing techniques have been developed for The DNA probes may also be used to identification of specific RNA sequences identify cells morphologically in specific (“Northern Blot”) and protein bands disease processes using “in-situ” hybrid (“Western Blot”) to allow the identifica ization techniques. In this approach, tion of mRNA and protein product whole cells or sections of tissue are expression of particular DNA sequences. mounted on slides, and labeled DNA A further refinement of these proce probes are annealed to the DNA within dures, the DNA polymerase technique, target cells. After development by auto promises to extend the sensitivity for radiography or immunochemistry, those detection of the abnormal DNA to a sin 142 BARROWS gle gene pair. In this method, a single ities within the discipline of hematology target area in the gene is amplified by are the result of abnormal genetic use of a bacterial DNA enzyme capable expression, parasitic infestation, or neo of amplifying single gene segments plastic change. As such, altered DNA between 20 and 1000 base pairs. The may play a significant role in the devel polymerase enzyme requires a primer opment of any of these disorders. In this pair which directs the start of DNA syn review, example applications will be thesis. After the cycle has been com used to illustrate potential uses of these pleted, the temperature of the mixture is agents in hematology (table I). Detailed raised converting the double stranded reviews of applications of these molecu DNA to single strands capable of repeat lar probes to neoplasia2 and hemoglobin ing the polymerase cycle. The tempera opathies9 are available which cover these ture is then lowered allowing polymer subjects in depth. ase to proceed, synthesizing an Cellular and humoral immunity are additional set of new chains from each of important lymphocytic functions for the complementary sequences pro maintenance of health. During lympho duced. The first synthetic products start cytic development a series of genetic at the target site proceeding from the 3' changes determine synthesis of charac end of the DNA to the 5' end producing teristic antibodies or cell surface recep its complementary sequence. After this, tors which direct the immune response repeat syntheses may take place starting against pathogens. Lymphocytic neo at the target site in the reverse direction plasms recapitulate features of normal of the targeted DNA on the recently syn development which may be recognized thesized complementary site. This pro by specific genetic probes and cell sur duces short chains which contain only face markers . 4 Cell surface markers, the specified site. Since each short seg however, may not be expressed in neo ment of DNA synthesized may serve as a plasms with disordered development. template for additional synthesis, expo nential growth of the reaction product occurs until the system is saturated. T A B L E I Using the polymerase technique, it is Desoxynucleic Acid Probe Applications to Hematology possible to amplify the number of Application Use Example selected gene pairs six million times. Historical documentation regarding Identification of Determination of Ig and T-cell genetic alteration cell lineage receptor the development of molecular probes is rearrangement well described in a recent publication Quantification of Detection of Ig and T-cell genetic expresión clonal expression receptor from the National Institutes of Health . 8 rearrangement

A recent review article about DNA tech Identification of Classification of Probe abnormal genetic disorders identification of nology is available which describes the material Philadelphia state of the art and provides an extensive chromosome reference listing for these techniques.5 Relapse Quantification of evaluation Philadelphia chromosome using polymerase Discussion techniques

Presence of Detection of foreign cells engrafted bone With modifications, these techniques marrow transplant have specificity and sensitivity which Identification of Documentation of Expression of promise to extend our diagnostic capabil gene products genetic activity Philadelphia identification chromosome mRNA ities in hematology. Nearly all abnormal APPLICATIONS OF DNA PROBES IN HEMATOLOGY 143

Genetic changes, on the other hand, are TABLE II always present as part of the develop Lineages of Lymphocytic Neoplasms mental process. The presence of DNA markers of specialized lymphocyte L in e a g e Di s o r d e r development are becoming important in categorizing and selecting treatment T cell derived: Acute lymphocytic leukemia Lymphoblastic lymphoma protocols for lymphoma and leukemia. Mycosis fungoides Large cell non-Hodgkins lymphoma During B-cell lymphocyte develop Immunoblastic sarcoma, T-cell type ment of humoral immunity, rearrange Lennert's lymphoma Angioimmunoblastic lymphadenopathy ments of the segments of DNA which encode heavy immunoglobulin (Ig) B cell derived: Acute lymphocytic leukemia Hairy cell leukemia chains occur. These changes are followed Follicular lymphoma Chronic lymphocytic leukemia by a sequence of changes for specific Burkitt's lymphoma light chains immunoglobulins. 6 Similar Large cell non-Hodgkins lymphoma changes occur in the DNA of T-Cells at Immunoblastic sarcoma the T-Cell Receptor (TCR) locus during Non lymphocyte : Acute lymphoblastic transformation of chronic myelogenous leukemia T-Cell development. The normal func tion of these changes is to allow specific identification of target sites from anti genic determinants, enabling the devel in this area has been difficult leading to a opment of immunologic competence. In diagnosis of “pseudolymphoma” with an normal development, Ig and TCR rear uncertain prognosis. The authors of this rangements are polyclonally expressed study used two restriction endonucleases and the numbers of cells with identical and hybridization of a DNA probe to the changes are below the three to five per “j” or joining region immunoglobulin cent frequency necessary for detection. heavy chains. This allowed identification Since neoplastic changes are clonal, spe of clonal rearrangements in two lympho cific rearrangements in genes are cytic proliferations which otherwise demonstrable in a large number of the would have been classified as benign affected cells giving rise to distinct processes. The presence of the B clonal markers for clonal neoplastic develop rearrangements and absence of T Cell ment. If the morphologic evidence for receptor rearrangements in these speci malignancy is equivocal, the presence of mens clearly documented neoplastic and clonal Ig or TCR rearrangements may be probable malignant change of B cell decisive factors in allowing a malignant type. diagnosis. The detection of these Specific genetic alterations associated markers also helps to classify lymphocy with hematological neoplasia can now be tic malignancies by providing objective readily identified by specific DNA evidence of their lineage and develop probes (table III). The application of mental status.4 these techniques for detection of genetic The use of these techniques with sub- changes associated with chronic myelog types of lymphocytic neoplasms allows enous leukemia provides insight into them to be categorized into will estab future routine applications for genetic lished groups (table II). An example of probes. For many years, the characteris useful application of gene rearrange tic 9;22 translocation has been known to ments was illustrated in a study of ocular be associated with 89 percent of cases of adnexal lymphoid neoplasms. 7 In prac chronic myelogenous leukemia. Using tice, the separation of malignant (clonal) specific probes, alterations in the active proliferations from reactive hyperplasia site where translocation breaks occur can 144 BARROWS TABLE III detection of mRNA from the 9;22 chro Examples of Specific Chromosomal Breakpoint mosome translocation present in patients Sites in Hematologic Neoplasms who have had lymphocytic transforma

G e n e tic tion of chronic myelogenous leukemia. 1 Diseases Translocation A lte r a tio n Chronic myelogenous leukemia may ter minate in a lymphoblastic crisis which is Chromic t(9;22) c-ahl (9) myelogenous bcr(22) morphologically indistinguishable from leukemia acute lymphocytic leukemia. The inves B cell lymphoma/ t(ll;14) Ig HC(14) tigators in this study used Northern Blot leukemia bcl- 1 (1 1 ) methods to demonstrate the characteris Follicular t{14;18) Ig HC (14) tic mRNA fragments complementary to lymphoma bcl-2 (18) the 9;22 translocation of chronic myelog Spordaic Burkits t(8,14) Ig HC(14) lymphoma t (8 ; 22) Lambda(22) enous leukemia. In this way, the origin t(8 ;2) Kappa(2) c-myc(8) of this disorder was demonstrated to be chronic myelogenous leukemia despite T-acute t(ll;14) TCR alpha Chain(14) lymphocytic its lymphocytic morphology. leukemia Deletions in DNA sequences may also be associated with malignancy. The detection of these deletions may be very useful in establishing a malignant or pre- be detected .2 This region is referred to malignant disease in cases where the as the bcr or “break point cluster diagnosis is equivocal. For example, region”. When molecular probes deletions are detectable in myeloid dys directed to this region are used, 95 per plasia using Southern Blotting tech cent of cases of chronic myelogenous niques with specific probes. 2 This evi leukemia have demonstrable changes. dence may be important in establishing Using the DNA polymerase technique, the diagnosis of myeloid dysplasia, a pre- chromosomal translocations such as malignant condition, since the hemato those occurring with chronic myeloge logic findings are often equivocal. nous leukemia can be selectively ampli Although probes for specific genetic fied by a probe directed toward the alterations are desirable, molecular translocated 9;22 sequence. Since nor applications which allow identification of mal cells will lack this sequence, this general DNA alterations using restric method promises to provide improved tion fragment label polymorphism or sensitivity for the presence of the t(9;22) “RFLPs” may be used in lieu of specific translocation. An estimate of the number genetic markers. A useful application of of abnormal cells expressed can thus be RFLP detection has been the documen obtained in patients who do not have tation of altered DNA present indicating morphologic evidence of disease. With successful engraftment of bone marrow this application, the polymerase tech donor cells. 2 nique is sufficiently sensitive to allow the extension of DNA probe methodology to Summary its theoretical limit of one gene pair. In addition to detection using DNA The future application for DNA probes, the presence of the Philadelphia probes in hematology is particularly chromosome can be verified indirectly promising since the majority of disorders by looking for the expression of the 9;22 are neoplastic or genetic. The tech translocation region in characteristic niques illustrated here exemplify the mRNA sequences. An elegant example expanse of the methods available. Given of the use of gene expression is the the specific nature of these probes and APPLICATIONS OF DNA PROBES IN HEMATOLOGY 145 their increasing commercial availability, templates (the reverse of normal it seems probable that they will be an DNA to RNA transcription). important part of the practice of labora Southern Blot-A DNA identification tory medicine in the future. process developed by Dr. Southern which involves separation DNA fragments by gel electrophoresis fol lowed by blot transfer to a medium Glossary of Term s allowing DNA hybridization and identification specific bands. Blot Transfer-A method of transferring T Cell Receptor Rearrangement—Char separated DNA, RNA or protein acteristic rearrangements which segments by absorption from gel occur in the DNA of lymphocytes medium to medium suitably stable during development of cellular specific band identification. immunity. Clonal Molecular Rearrangements - Western Blot-A blot transfer technique Characteristic repetitive DNA rear using protein fragment gel electro rangements which occur during phoresis separation followed by blot selective growth of cells with identi transfer to a medium allowing iden cal DNA structures (clones). tification specific protein bands. DNA Hybridization—Binding of DNA markers to characteristic sites by References

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