10TH ANNIVERSARY ARTICLE Gene Mapping in Cancer Linda A. Cannizzaro
ABSTRACT: Observation of genetic alterations that appear consistently in specific types and stages of cancer provides a strong impetus to cancer geneticists to fl~cus their investigations on the exploration of such volatile regions of the h u m a n genome. Introduction of powerful malecular cytogenetic and molecular genetic methods in recent years permits more detailed analysis, which will help researchers in their efforts to determine if such areas of the human genome have a functional role in the initiation and progressive development of leukemias and solid tumors. This discussion will focus on several provocative molecular cytogenetic tools that are currently available to localize potential cancer-assoeiated genes and on how these methods are being used in conjunction with the current modes of analysis, including cytogenetics and somatic cell genetics. In addition, we will explore how these methods will help to isolate and dissect recently discovered cancer-associated genes within the human genome. All o) these methods used in combination with each other will provide essential DNA markers fl)r future diagnostic and prognostic evaluation of cancer.
INTRODUCTION It w o u l d be an exceptional a c h i e v e m e n t if a cancer geneticist was able to resolve subtle alterations in a patient's DNA to distinguish an i n d i v i d u a l with progressive disease from an i n d i v i d u a l at a less advanced stage of malignant d e v e l o p m e n t . The recent introduction of several powerful m o l ecu l ar cytogenetic tools is making this a c h i e v e m e n t an eventual possibility. The use of these n ew methods allows more detailed inspection and finer resolution of subtle cytogenetic and m o l ecu l ar alterations in leukemias and solid tumors. These exciting new d e v e l o p m e n t s are aiding the cancer geneticist's ability to target precise areas of the h u m a n genome that may contain cancer-associated gene loci. Such genetic loci most likely contribute to the initiation, d e v e l o p m e n t , and c o n ti n u e d progression of a particular malignancy. The cancer geneticists' research is starting to become more fruitful and productive as a result of the vastly i m p r o v e d technical resources currently available to them.
Mapping Cancer-Associated Genes With Cytogenetics Major breakthroughs in high-resolution banding have e n o r m o u s l y e n h a n c e d the cytogenetic interpretation of subtle c h r o m o s o m e alterations in patients with cancer. As a result, only a limited n u m b e r of c h r o m o s o m e bands have been implicated in cancerassociated abnormalities [40-43, 49-511. These alterations are manifested either as deletions, duplications, translocations, or inversions of genetic material. A significant
From The Fels Institute for Cancer Research and Molecular Biology,Temple University School of Medicine, Philadelphia. Pennsylvania. Address reprint requests to: Linda A. Cannizzaro, PhD., Jefferson Cancer Institute, Jefferson Medical College, A l u m n i Hall, 1020 Lacust Street, Philadelphia, PA 19107. Received December 4, 1990; accepted January 31, 1991.
139 c~.t1991 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10010
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effort has been undertaken by laboratories around the world to identify consistent alterations in association with specific malignancies. These efforts have been compiled from a diversity of leukemias and solid tumor specimens [26, 50]. The results of these investigations reveal that at least 149 n o n r a n d o m c h r o m o s o m e changes have been identified in at least 43 different malignancies, with the majority of the changes occurring in the leukemias [26]. Only 20% of the defined cytogenetic alterations are associated with solid tumors. However, this indicates significant i m p r o v e m e n t in the ability to detect abnormalities in these tumors, because they are so difficult to culture in vitro. Cytogenetic evaluation is important in the current effort now u n d e r w a y to map genes involved in the initiation, development, and progression of cancer. Alterations that involve certain c h r o m o s o m e regions consistently provide clues as to what areas of the h u m a n genome are more susceptible to mutagenic events. These areas can then be carefully scrutinized using a plethora of molecular analytic methods. Some examples i n c l u d e the c h r o m o s o m e translocations in patients with Burkitt l y m p h o m a in which the c-myc oncogene located in 8q24 is consistently translocated into close proximity to a gene locus that codes for an i m m u n o g l o b u l i n chain [9-13, 18, 19, 24, 25, 36, 37, 46, 47, 54[. Identification by cytogenetic analysis of a region consistently altered in association with a type or stage of malignancy provides the basis for determining whether or not a inolecular alteration may have taken place at a corres p o n d i n g or nearby locus along the chromosome. In the case of the c-myc oncogene, aberrant expression is consistently observed when it becomes reoriented near an i m m u n o g l o b u l i n gene. The breakpoint region in such cases can be used to p i n p o i n t the location of other genes on the derivative c h r o m o s o m e by serving as a genetic marker that defines one subregion from another along the same c h r o m o s o m e and, in some cases, within the same chroinosome band. In cases where deletions of a chromosoIne region are consistently associated with a type or stage of malignancy, they provide essential information about the role such sequences may have in i n d i v i d u a l s who do not manifest the disease. For instance, initial cytogenetic screening of m e l a n o m a tumors demonstrate consistent absence of sequences from c h r o m o s o m e 6 [57]. If this c h r o m o s o m e is replaced in these tumor cells, there is a concomitant loss of tumorigenicity when injected into n u d e mice [57}. This discovery uncovers the location of potential suppressor loci along c h r o m o s o m e 6, w h i c h may be an important factor in ultimately treating patients with melanoma. Similar discoveries have been uncovered in an inherited tumor, retinoblastoma [2]. Patients are initially diagnosed cytogenetically by the observation of a deletion in the 13q14 locus [501. Subsequent investigations have found that suppressor loci are located within this c h r o m o s o m e region [21. Such findings would not have been possible without p r e l i m i n a r y indications from cytogenetic analyses. Knowledge regarding the location of such tumor suppressor loci will hopefully lead to the eventual isolation and characterization of corresponding loci from normal individuals, w h i c h w o u l d then pave the way for generating appropriate diagnostic markers for each malignancy. Such findings may also have important value to potential gene therapeutic regimens in inherited cancers.
Mapping Cancer-Associated Genes by Somatic Cell Hybridization Somatic cell h y b r i d i z a t i o n is all important tool that c o m p l e m e n t s both cytogenetic and in situ h y b r i d i z a t i o n methods to map genes associated with neoplasia. The hybrid ceils are generated by the fusion of somatic cells of two different parental species, usually rodent and h u m a n in selective HAT (hypoxanthine a m i n o p t e r i n thymidine) m e d i u m [601. In the resulting hybrids, all the rodent chromosomes are retained, while the h u m a n c h r o m o s o m e s are lost, d e p e n d i n g on w h i c h selective c o m p o n e n t s are a d d e d to the m e d i u m [11,60]. The hybrid panels selectively retain one or more h u m a n
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c h r o m o s o m e s and are used to localize genes to either whole c h r o m o s o m e s or specific regions of chromosomes, d e p e n d i n g on their cosegregation with k n o w n DNA markers. Somatic cell hybrids have become extremely important in recent years in m a p p i n g cancer-associated genes. Hybrid panels have been constructed from transformed cells w h i c h contain c h r o m o s o m e alterations [11] that originate from patients with malignancies at different stages of malignant progression. Hybrids that retain marker chromosomes can then be used in a variety of ways. In most cases, it is possible to localize a DNA probe to a c h r o m o s o m e subregion, as well as to determine the relationship of genes along the altered c h r o m o s o m e segment using a series of hybrid panels. A n u m b e r of hybrid panels have been constructed from patients with Burkitt l y m p h o m a who carry c h r o m o s o m e alterations w h i c h involve the c - m y c oncogene located at 8q24.1 in exchange with segments of chromosomes 2, 14. and 22 in the form of a c h r o m o s o m e translocation event [12, 18, 19, 24, 47]. These hybrids are being used to identify and localize DNA probes that are nearby or intercept the translocatioo breakpoint region of each derivative c h r o m o s o m e retained in the panels. The sequential ordering of genes can be achieved using panels that contain different translocations of the same c h r o m o s o m e region [11]. The location of the i m m u n o g l o b u l i n chain gene could be defined within the same vicinity of the myc oncogene in a series of hybrids w h i c h contained the derivative translocation chromosome as the sole h u m a n element [10, 11, 12, 24, 25]. Two modified strategies have also been useful to order cancer-associated genes with somatic cell hybrid panels. The first is c h r o m o s o m e - m e d i a t e d gene transfer [391: the second method involves irradiation-fusion [21, 22]. C h r o m o s o m e - m e d i a t e d gene transfer involves the inclusion of c h r o m o s o m e fragments into recipient cells as a calcium p h o s p h a t e precipitant. This method has successfully ordered a number of gene loci, i n c l u d i n g several oncogenes [21]. However, it is still not k n o w n w h e t h e r the selected c h r o m o s o m e fragments m a i n t a i n their organizational integrity and could easily become rearranged in the resultant hybrids [1]. The irradiation fusion method is believed to have more potential for the recent h u m a n genome m a p p i n g initiative and can be used to map very small subchromosoreal fragments less than 10,000 kb in size [23]. Even smaller fragments can be selected for in hybrids already irradiation reduced that contain very few or only one h u m a n chromosome [7, 8, 21]. Such hybrids are generated by fragmenting the c h r o m o s o m e s of one parent with gamma radiation prior to fusion [22, 60]. Use of this method has recently resulted in the isolation of c h r o m o s o m e subfragments from 11p, which span the region containing the genes believed responsible for a n i r i d i a - W i l m s ' tumor in 11p13 [21]. These hybrids retain only small pieces of the 11p13 region and thus provide a m e c h a n i s m that will facilitate the isolation of the h u m a n sequences responsible for Wilms tumor [21]. Somatic cell hybrids have not only been informative in d e t e r m i n i n g whether the presence or absence of a specific c h r o m o s o m e can be associated with a certain type or stage of malignancy, they have been very helpful in targeting defined c h r o m o s o m e regions responsible for the altered transcription of a protein product. The altered p r o d u c t i o n of myc protein products has been found only in those hybrids in w h i c h the myc oncogene is reoriented near i m m u n o g l o b u l i n sequences [18, 19]. Correlations such as these are valuable diagnostic tools to distinguish patients with very defined clinical parameters of a specific type or stage of cancer.
Mapping Genes Associated with Cancer by In Situ Hybridization M a p p i n g and ordering cancer-associated genes by somatic cell h y b r i d i z a t i o n alone d e p e n d s on the ability to generate a sufficient n u m b e r of clones that retain different segments of the same c h r o m o s o m e or set of chromosomes. Because of the extensive c h r o m o s o m e instability and constant rearrangement between the h u m a n and rodent
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elements, this is often extremely difficult to accomplish. In addition, such hybrid panels often require constant monitoring and screening to ensure they contain the h u m a n elements that were selected for initially. Chromosomal in situ h y b r i d i z a t i o n localizes a DNA sequence directly to a precisely defined c h r o m o s o m e segment or band. The h y b r i d i z a t i o n signal is detected either with radioactively labeled (usually with :~H) DNA probes or probes labeled with nonradioactive c o m p o u n d s such as biotin 11-dUTP. The hybridization signal from nonradioactive labeled probes can then be detected with a variety of immunofluorescent c o m p o u n d s such as fluorescein (FITC) conjugated with avidin. In situ h y b r i d i z a t i o n has been used in conjunction with somatic cell h y b r i d i z a t i o n to regionally localize genes less than 0.5 kb to a precisely defined c h r o m o s o m e locus. 'The in situ technique permits visualization of genetic alterations associated with cancer at a m u c h higher level of resolution than cytogenetics or somatic cell hybridization. A series of probes that map to a c h r o m o s o m e region involved in a variety of malignancies will result in the construction of a physical map in w h i c h the order of genes can be ascertained in relation to the altered chromosome segment. For instance, using several probes derived from the l a m b d a light chain i m m u n o g l o b u l i n locus at 22q11, it was possible to determine the location of genes potentially responsible for the manifestation of several constitutional and malignant disorders in w h i c h c h r o m o s o m e 22 participated in a chromosome translocation event. These i n c l u d e d defining the breakpoint region of a 4:22 translocation observed in a patient with DiGeorge s y n d r o m e [4]: defining the breakpoint of the 9:22 translocation diagnostic of chronic myelogenous leukemia (CML) [10, 17]; defining the breakpoint of the 8;22 translocation characteristic of patients with variant Burkitt l y m p h o m a [16]; defining the sequences involved in a patient diagnosed with acute l y m p h o c y t i c leukemia (ALL) with a 9;22 rearrangement [5, 10]. In each case, the l a m b d a gene h e l p e d to define the order of translocation breakpoints in relation to each other and also h e l p e d to target specific sequences along c h r o m o s o m e 22 as potential sites that may contain cancer-associated loci. Four different probes that define breakpoint cluster region (bcr) loci have been m a p p e d in relation to each other as well as to the cancer breakpoint of both ALL and CML [10], using both in situ h y b r i d i z a t i o n and somatic cell hybridization. In some cases, in situ hybridization, especially with radioactively labeled probes, will detect alterations of a gene that cannot be defined cytogenetically. For example, in the ML-3 cell line established from a patient with acute myelogenous leukemia, there was no visible rearrangement of sequences from chromosome 22 to c h r o m o s o m e 17. However, in situ h y b r i d i z a t i o n with a c-sis oncogene probe that maps to the 22q13 region detected a reorientation of c h r o m o s o m e 22 sequences to the long arm of c h r o m o s o m e 17 [58]. In addition, it is possible to use in situ h y b r i d i z a t i o n to quantitate the presence or absence of sequences along a chromosome, w h i c h in some malignancies is manifested either as d u p l i c a t i o n s or deletions of a c h r o m o s o m e locus. Numerous other examples can be cited. In situ hybridization, therefore, has become an essential tool to define sequences or genes involved in a malignancy, and is being used to construct a physical map of genes along the c h r o m o s o m e in relation to other k n o w n DNA markers and cancer breakpoint regions. Maps generated in this way for c h r o m o s o m e regions consistently involved in neoplasia have increased the resolution of these areas of the h u m a n genome previously unattainable by cytogenetic evaluation alone. The m a p p i n g potential of in situ h y b r i d i z a t i o n technology has e x p a n d e d considerably as a result of the increased use of non-radioactive immunofluorescent c o m p o u n d s such as FITC conjugated to avidin [31-34, 44, 45, 55]. Recent i m p r o v e m e n t s in this technique now permit detection of signal from unique DNA probes that range in size from 0.2-0.5 kb in length as a result of repeated amplification with FITC [34]. The use of immunofluorescent c o m p o u n d s has significant advantages, especially for clinical
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diagnosis of malignant disorders where it is not always possible to generate analyzable metaphases. Unlike the radioactive in situ c o m p o u n d s , it is not necessary to expose t h e m for e x t e n d e d periods over an autoradiographic e m u l s i o n [31-34, 44, 451. In most cases, the preparations are m u c h cleaner with significantly less background than radioactive preparations. This enhances the overall quality and facilitates quantitation of signal over specific c h r o m o s o m e areas. One advantage that the radioactive detection m e t h o d still retains, however, is its ability to detect signal from very small DNA fragments less than 500 bp. A significantly larger quantity of DNA is necessary for detection of such small unique sequences with the non-radioactive method, and requires repeated amplifications with FITC to c o m p e n s a t e for this difficulty. In laboratories performing diagnostic evaluation of tumor-derived samples, it is important to maintain flexibility in establishing the c h r o m o s o m a l in situ h y b r i d i z a t i o n procedures, so that it is possible to switch from one to the other d e p e n d i n g on the tumor s a m p l e and DNA probe being used to evaluate a chromosomal alteration. Non-radioactive detection also has the ability to detect numerical and structural c h r o m o s o m e alterations in the interphase stage as well as in the m e t a p h a s e stage. In some tumors, such as b l a d d e r cancer, it is difficult to establish biopsy samples in vitro for e x t e n d e d periods and results in an insufficient number of analyzable metaphases (Sandberg, personal communication). Detection of c h r o m o s o m e abnormalities in interphase can be achieved in several ways. The most c o m m o n method involves the use of a chromosome-specific flow-sorted library [31,44, 45]. This results in " c h r o m o s o m e painting" and has been used to detect numerical aberrations such as trisomy 21 [44, 45], and a 4',11 translocation in a leukemia cell line [45]. In metaphases, the use of total h u m a n genomic DNA labeled with biotin has facilitated rapid screening of h u m a n - r o d e n t hybrid panels. In such hybrids, it is possible to detect as little as 1 × 107 bp of h u m a n DNA inserted into a rodent c h r o m o s o m e [15, 45]. More recent a p p l i c a t i o n s of fluorescent in situ h y b r i d i z a t i o n include detecting signal from m u l t i p l e probes either in metaphase or in the interphase cell [28-30, 55, 59]. In one case, it was possible to detect numerical and structural alterations of c h r o m o s o m e 1 in the leukemia cell line K562 using a combination of telomeric and centromeric probes each labeled with biotin and h y b r i d i z e d at differing concentrations [59]. In addition, studies with m u l t i p l e fluorophores now permit localization of several probes w i t h i n the same metaphase or interphase [28, 29, 55,561. This is of pivotal i m p o r t a n c e in clinical diagnosis, where both the linear relationship and organization of genes can be readily defined with probes that fluoresce different colors due to their activation at differing ultraviolet wavelengths. For example, in patients diagnosed with Ph + CML, it is possible to detect the 9q + c h r o m o s o m e by dual hybridization with bcr and abl probes each labeled with a different fluorophore [55]. W h e n counterstained, the fusion of the bcr-abl sequences was discerned as two distinctly colored fluorescent signals along the same c h r o m o s o m e [55]. In addition, m u l t i p l e copies of the translocation chromosome could be detected in the interphase stage. It is now possible to visualize fluorescent signals over chromosomes b a n d e d with c o m p o u n d s like DAPI and Hoechst 33258, w h i c h will elicit bands similar to G- or Rbands, respectively [6, 20]. The origin of c h r o m o s o m e s involved in cancer-associated numerical and structural abnormalities can thus be more precisely identified. The c o m b i n e d use of the radioactive and fluorescent in situ h y b r i d i z a t i o n method will provide an effective strategy to define numerical and structural alterations in all malignant types and stages.
Isolation of Sequences Consistently Altered in Neoplasia by Chromosome Microdissection Microdissection of h u m a n c h r o m o s o m e s is a relatively new and powerful tool in w h i c h DNA " m i c r o c l o n e s " are directly dissected from a c h r o m o s o m e region by m i c r o m a n i p u l a t i o n . The first microclones were derived from the m i c r o d i s s e c t i o n ot
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salivary gland polytene chromosomes of Diptera [53]. Such clones were isolated without any special treatment to distinguish a specific chromosome or band region. The chromosomes of interest, especially translocations, were thus identified on the basis of morphology alone [48]. More recently, microdissection is performed on fixed high-resolution banded chromosomes [3, 27, 35, 38, 52, 61]. DNA is dissected from a chromosome band or subregion that is precisely defined and thus the creation of DNA libraries from an identifiable target sequence along the chromosome is facilitated. The microdissection technique involves moving a fine needle, usually 0.1-0.5/xm in diameter, with a micromanipulator. DNA fragments are dissected from varying n u m b e r s of metaphases, from a few as 6 to as many as 50. The dissected fragments are pooled together and digested with a restriction enzyme, such as Rsal, which yields very small fragments. These fragments range in size from 50-350 bp [351 and when cut with other restriction enzymes, fragments up to 11 kbp can be generated {27]. The advantage of isolating such small DNA fragments is that the majority of them, about 80%, will be u n i q u e sequence clones without linked repetitive sequences [35l. This is a significant benefit, especially in isolating regions that most likely contain actively transcribed genes and disease-associated loci. These fragments can be inserted into an appropriate vector system and then amplified by the polymerase chain reaction (PCR) for further molecular characterization. The use of microdissection to isolate the genes involved in chromosome alterations in leukemia and solid tumors will provide a rapid means of creating DNA libraries from these altered regions. Libraries have already been generated from patients with neuroblastoma who manifest consistent alterations of the short arm of chromosome 1 [38]. The DNA clones isolated by chromosome microdissection can then serve as genetic markers or detect restriction fragment-length polymorphisms (RFLPs) that will identify at-risk or carrier individuals. Similar strategies have been undertaken to isolate clones from within the 11p13 chromosome region that contains the genes responsible for manifestation of the aniridia Wilms' tumor complex [14]. Multiple clones can be isolated and used to construct physical maps of these cancer-associated regions of the h u m a n genome. The u n i q u e sequence clones generated from microdissection are used as start points to pull out adjacent sequences along the chromosome, similar to the more conventional approach of walking along the chromosome. Once the precise location of each clone is confirmed either by in situ hybridization or somatic cell hybridization, they can be sequenced and compared to homologous segments from i n d i v i d u a l s who do not manifest any disease. Characterization ot cancer-associated altered DNA in this way will be essential at differing stages of the disease progression to help define the association of a specific stage of malignant progression with the manifestation of a cytogenetic or molecular abnormality. Ultimately, this fine dissection of the h u m a n genome will provide the necessary diagnostic markers to finally discern specific tumor types and stages of malignant progression. In summary, a n u m b e r of tools are now available to map cancer-associated genes. Used in combination with each other, they provide very powerful means to dissect vulnerable areas of the h u m a n genome that are responsible for the manifestation and progression of neoplasia. REFERENCES
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25. Hollis GF, Mitchell KF, Battey J, Potter H, Taub R, Lenoir GM, Leder P (1984): A variant translocation places the lambda immunoglobulin genes 3' to the c-myc oncogene in Burkitt lymphoma. Nature 307:752-755. 26. Human Gene Mapping 10 (1989): New Haven Conference, 10th International Workshop on human gene mapping. Cytogenetics Cell Genet 51:533-562. 27. Johnson DH (1990): Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA. Genomics 6:243-251. 28. Lawrence JB, Marselle LM, Byron KS, Johnson CV, Sullivan JL, Singer RH (1990a): Subcellular localization of low abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization. Proc Natl Acad Sci USA 87:5420-5424. 29. Lawrence JB, Singer RH, McNeil JA (1990b): interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249:928-932. 30. Lawrence ]B, Villnove CA, Singer RH (1988): Sensitive, high resolution chromatin and chromosomal mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 32:51 61. 31. Lichter P, Cremer T, Tang C-JC, Watkins PC, Manuelidis L, Ward DC (1988): Rapid detection of human chromosome 21 aberrations by in situ hybridizations. Proc Natl Acad Sci USA 85:9664-9668. 32. Lichter P, Ledbetter SA, Ledbetter DH, Ward DC (1990): Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid (:ell lines. Proc Natl Acad Sci USA 87:6634-6638. 33. Lichter P, Tang C-JC, Call K, Hermanson G, Evans GA, Housman D, Ward DC (1990): Highresolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Sciellc:e 247:64-69. 34. Lichter P, Ward DC (1990): is non-isotopic in situ hybridization finally coming of age? Nature 345:93-94. 35. Ludecke H-J, Senger G, Claussen U, Horsthemke B (1989): Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348-350. 36. Malcohn S, Davis M, Rabbitts TH (1985): Breakage on chromosome 2 brings the C-K gene to a region 3' of c-myc in a Burkitt lymphoma line carrying a (2;8) translocation. Cytogenet Cell Genet 39:168-172. 37. Manolov G, Manolova Y, Klein G, Lenoir G, Levan A (1986): Alternative involvement of two cytogenetically distinguishable breakpoints on chromosome 8 in Burkitt lymphoma associated translocations. Cancer Genet Cytogenet 20:95-99. 38. Martinsson T, Weith A, Cziepluck C, Schwab M (1989): Chromosome 1 deletions in human neuroblastoma: Generation and fine mapping of microclones from the distal l p region. Genes, Chromosomes and Canker 1:67--78. 39. McBride OW, Ozer HL (19731: Transfer of genetic information by purified metaphase chromosomes. Proc Natl Acad Sci USA 70:1258-1262. 40. Mitehnan F (1984): Restricted number of chromosomal regions implicated in aetiology oI human cancer and leukemia. Nature 310:325-327. 41. Mitelman F (1986a): (;lustering of chromosomal breakpoints in neoplasia. Cancer Genet Cytogenet 19:67-71. 42. Mitelman F (1986b): Clustering of breakpoints to specific chromosomal regions in human neoplasia: A survey of 5,345 cases. Hereditas 104:113-119. 43. Mitehnan F, Levan G (1981): Clustering of aberrations to specific: chromosomes in human neoplasms IV. A survey of 1,871 cases. Hereditas 95:79-139. 44. Moyzis RK, Albright KL, Bartholdi MF, Cram LS, Deaven LL, Heldebrand CE, Joste NE, Longmire JL, Meyne J, Schwarzacher-Robinson T (1987): Human chronmsome-specific repetitive DNA sequences: Novel markers for genetic analysis. Chromosoma 95:375-386. 45. Pinkel D, Landegent J, Collins C, Frescal J, Segraves R, Lucas J, Gray J (1988): Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocation of chromosome 4. Proc Natl Acad Sci USA 85:9138-9142. 46. Rabbitts TH, Forster A, Hamlyn P, Beer R (1984): Effect of somatic mutation within translocared c-myc genes in Burkitt lymphoma. Nature 309:592-597. 47. Rappold GA, Hameister H, Cremer T, Adolph S, Henglein B, Freese U-K. Lenoire GM,
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Bornkamm GW {1984): c-myc and immunoglobulin K light chain constant genes are on the 8q+ chromosome of three Burkitt lymphoma lines with t(2;8) translocations. EMBO J 3:2951-2955. Rohme D, Fox H, Herrmann B, Frischauf AM, Edstrom I-E, Mains P, Silver LM, Lehrach H (1984): Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes. Cell 36:783-788. Rowley JD (1984): Biological implications of consistent chromosome rearrangements in leukemia and lymphoma. Cancer Res 44:3159-3168. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia, Second Edition, Elsevier Science Publishing Co., New York, N.Y. Sandberg AA, Turc-Carel C, Gemmill RM (1988): Chromosomes in solid tumors and beyond. Cancer Res 48:1049-1059. Saunders RDC, Glover DM, Ashburner M, Siden-Kiamos I, Louis C, Monastriati M, Savakis C, Kafatos F (1989): PCR amplification of DNA microdissected from a single polytene chromosome band: a comparison with conventional microcloning. Nucl Acids Res 17:9027-9037.
53. Scalenghe F, Turco E, Edstrom JE, Pirotta V, Melli M (1981/: Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes. Chromosoma 82:205-216. 54. Showe LC, Moore RCA, Erikson J, Croce CM (1987/: Myc oncogene involved in a t(8:22) chromosome translocation is not altered in its putative regulatory regions. Pro(: Natl Acad Sci USA 84:2824-2828. 55. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Redner A, Gray J, Pinkel D (1990): Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization. Science 250:559 562. 56. Trask B, Pinkel D, Van den Engh G (1989): The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5 : 7 1 0 - 7 1 7 . 57. Trent JM, Stanbridge E l, McBride HL, Meese EU, Casey G, Araujo DE, Witkowski CM, Nagle RB (1990): Tumorigenicity in h u m a n melanoma cell lines controlled by introduction of h u m a n chromosome 6. Science 247:568-571. 58. Tripputi P, Cannizzaro LA, Lindan C, Kuo J, Letovsky J, Klepfer S, Caputo A, Corneo G (1987): Translocation of oncogene c-sis from chromosome 22 to chromosome 17 in a h u m a n myelogenous leukemia cell line. Acta Haemat 77:135-139. 59. Van Dekken H, Bauman JGJ (1988): A new application of in situ hybridization: detection of numerical and structural chromosome aberrations with a combination centromerictelomeric DNA probe. Cytogenet Cell Genet 48:188-189. 60. Weiss MC, Green H (1967): Human-mouse hybrid cell line containing partial complements of h u m a n chromosomes and functioning h u m a n genes. Proc Natl Acad Sci USA 58:1104-1111. 61. Wesley CS, Ben M, Kreitman M, Hagag N, Eaves WF (1990): Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer. Nucl Acids Res 18:599-603.
ABSTRACT: Observation of genetic alterations that appear consistently in specific types and stages of cancer provides a strong impetus to cancer geneticists to fl~cus their investigations on the exploration of such volatile regions of the h u m a n genome. Introduction of powerful malecular cytogenetic and molecular genetic methods in recent years permits more detailed analysis, which will help researchers in their efforts to determine if such areas of the human genome have a functional role in the initiation and progressive development of leukemias and solid tumors. This discussion will focus on several provocative molecular cytogenetic tools that are currently available to localize potential cancer-assoeiated genes and on how these methods are being used in conjunction with the current modes of analysis, including cytogenetics and somatic cell genetics. In addition, we will explore how these methods will help to isolate and dissect recently discovered cancer-associated genes within the human genome. All o) these methods used in combination with each other will provide essential DNA markers fl)r future diagnostic and prognostic evaluation of cancer.
INTRODUCTION It w o u l d be an exceptional a c h i e v e m e n t if a cancer geneticist was able to resolve subtle alterations in a patient's DNA to distinguish an i n d i v i d u a l with progressive disease from an i n d i v i d u a l at a less advanced stage of malignant d e v e l o p m e n t . The recent introduction of several powerful m o l ecu l ar cytogenetic tools is making this a c h i e v e m e n t an eventual possibility. The use of these n ew methods allows more detailed inspection and finer resolution of subtle cytogenetic and m o l ecu l ar alterations in leukemias and solid tumors. These exciting new d e v e l o p m e n t s are aiding the cancer geneticist's ability to target precise areas of the h u m a n genome that may contain cancer-associated gene loci. Such genetic loci most likely contribute to the initiation, d e v e l o p m e n t , and c o n ti n u e d progression of a particular malignancy. The cancer geneticists' research is starting to become more fruitful and productive as a result of the vastly i m p r o v e d technical resources currently available to them.
Mapping Cancer-Associated Genes With Cytogenetics Major breakthroughs in high-resolution banding have e n o r m o u s l y e n h a n c e d the cytogenetic interpretation of subtle c h r o m o s o m e alterations in patients with cancer. As a result, only a limited n u m b e r of c h r o m o s o m e bands have been implicated in cancerassociated abnormalities [40-43, 49-511. These alterations are manifested either as deletions, duplications, translocations, or inversions of genetic material. A significant
From The Fels Institute for Cancer Research and Molecular Biology,Temple University School of Medicine, Philadelphia. Pennsylvania. Address reprint requests to: Linda A. Cannizzaro, PhD., Jefferson Cancer Institute, Jefferson Medical College, A l u m n i Hall, 1020 Lacust Street, Philadelphia, PA 19107. Received December 4, 1990; accepted January 31, 1991.
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effort has been undertaken by laboratories around the world to identify consistent alterations in association with specific malignancies. These efforts have been compiled from a diversity of leukemias and solid tumor specimens [26, 50]. The results of these investigations reveal that at least 149 n o n r a n d o m c h r o m o s o m e changes have been identified in at least 43 different malignancies, with the majority of the changes occurring in the leukemias [26]. Only 20% of the defined cytogenetic alterations are associated with solid tumors. However, this indicates significant i m p r o v e m e n t in the ability to detect abnormalities in these tumors, because they are so difficult to culture in vitro. Cytogenetic evaluation is important in the current effort now u n d e r w a y to map genes involved in the initiation, development, and progression of cancer. Alterations that involve certain c h r o m o s o m e regions consistently provide clues as to what areas of the h u m a n genome are more susceptible to mutagenic events. These areas can then be carefully scrutinized using a plethora of molecular analytic methods. Some examples i n c l u d e the c h r o m o s o m e translocations in patients with Burkitt l y m p h o m a in which the c-myc oncogene located in 8q24 is consistently translocated into close proximity to a gene locus that codes for an i m m u n o g l o b u l i n chain [9-13, 18, 19, 24, 25, 36, 37, 46, 47, 54[. Identification by cytogenetic analysis of a region consistently altered in association with a type or stage of malignancy provides the basis for determining whether or not a inolecular alteration may have taken place at a corres p o n d i n g or nearby locus along the chromosome. In the case of the c-myc oncogene, aberrant expression is consistently observed when it becomes reoriented near an i m m u n o g l o b u l i n gene. The breakpoint region in such cases can be used to p i n p o i n t the location of other genes on the derivative c h r o m o s o m e by serving as a genetic marker that defines one subregion from another along the same c h r o m o s o m e and, in some cases, within the same chroinosome band. In cases where deletions of a chromosoIne region are consistently associated with a type or stage of malignancy, they provide essential information about the role such sequences may have in i n d i v i d u a l s who do not manifest the disease. For instance, initial cytogenetic screening of m e l a n o m a tumors demonstrate consistent absence of sequences from c h r o m o s o m e 6 [57]. If this c h r o m o s o m e is replaced in these tumor cells, there is a concomitant loss of tumorigenicity when injected into n u d e mice [57}. This discovery uncovers the location of potential suppressor loci along c h r o m o s o m e 6, w h i c h may be an important factor in ultimately treating patients with melanoma. Similar discoveries have been uncovered in an inherited tumor, retinoblastoma [2]. Patients are initially diagnosed cytogenetically by the observation of a deletion in the 13q14 locus [501. Subsequent investigations have found that suppressor loci are located within this c h r o m o s o m e region [21. Such findings would not have been possible without p r e l i m i n a r y indications from cytogenetic analyses. Knowledge regarding the location of such tumor suppressor loci will hopefully lead to the eventual isolation and characterization of corresponding loci from normal individuals, w h i c h w o u l d then pave the way for generating appropriate diagnostic markers for each malignancy. Such findings may also have important value to potential gene therapeutic regimens in inherited cancers.
Mapping Cancer-Associated Genes by Somatic Cell Hybridization Somatic cell h y b r i d i z a t i o n is all important tool that c o m p l e m e n t s both cytogenetic and in situ h y b r i d i z a t i o n methods to map genes associated with neoplasia. The hybrid ceils are generated by the fusion of somatic cells of two different parental species, usually rodent and h u m a n in selective HAT (hypoxanthine a m i n o p t e r i n thymidine) m e d i u m [601. In the resulting hybrids, all the rodent chromosomes are retained, while the h u m a n c h r o m o s o m e s are lost, d e p e n d i n g on w h i c h selective c o m p o n e n t s are a d d e d to the m e d i u m [11,60]. The hybrid panels selectively retain one or more h u m a n
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c h r o m o s o m e s and are used to localize genes to either whole c h r o m o s o m e s or specific regions of chromosomes, d e p e n d i n g on their cosegregation with k n o w n DNA markers. Somatic cell hybrids have become extremely important in recent years in m a p p i n g cancer-associated genes. Hybrid panels have been constructed from transformed cells w h i c h contain c h r o m o s o m e alterations [11] that originate from patients with malignancies at different stages of malignant progression. Hybrids that retain marker chromosomes can then be used in a variety of ways. In most cases, it is possible to localize a DNA probe to a c h r o m o s o m e subregion, as well as to determine the relationship of genes along the altered c h r o m o s o m e segment using a series of hybrid panels. A n u m b e r of hybrid panels have been constructed from patients with Burkitt l y m p h o m a who carry c h r o m o s o m e alterations w h i c h involve the c - m y c oncogene located at 8q24.1 in exchange with segments of chromosomes 2, 14. and 22 in the form of a c h r o m o s o m e translocation event [12, 18, 19, 24, 47]. These hybrids are being used to identify and localize DNA probes that are nearby or intercept the translocatioo breakpoint region of each derivative c h r o m o s o m e retained in the panels. The sequential ordering of genes can be achieved using panels that contain different translocations of the same c h r o m o s o m e region [11]. The location of the i m m u n o g l o b u l i n chain gene could be defined within the same vicinity of the myc oncogene in a series of hybrids w h i c h contained the derivative translocation chromosome as the sole h u m a n element [10, 11, 12, 24, 25]. Two modified strategies have also been useful to order cancer-associated genes with somatic cell hybrid panels. The first is c h r o m o s o m e - m e d i a t e d gene transfer [391: the second method involves irradiation-fusion [21, 22]. C h r o m o s o m e - m e d i a t e d gene transfer involves the inclusion of c h r o m o s o m e fragments into recipient cells as a calcium p h o s p h a t e precipitant. This method has successfully ordered a number of gene loci, i n c l u d i n g several oncogenes [21]. However, it is still not k n o w n w h e t h e r the selected c h r o m o s o m e fragments m a i n t a i n their organizational integrity and could easily become rearranged in the resultant hybrids [1]. The irradiation fusion method is believed to have more potential for the recent h u m a n genome m a p p i n g initiative and can be used to map very small subchromosoreal fragments less than 10,000 kb in size [23]. Even smaller fragments can be selected for in hybrids already irradiation reduced that contain very few or only one h u m a n chromosome [7, 8, 21]. Such hybrids are generated by fragmenting the c h r o m o s o m e s of one parent with gamma radiation prior to fusion [22, 60]. Use of this method has recently resulted in the isolation of c h r o m o s o m e subfragments from 11p, which span the region containing the genes believed responsible for a n i r i d i a - W i l m s ' tumor in 11p13 [21]. These hybrids retain only small pieces of the 11p13 region and thus provide a m e c h a n i s m that will facilitate the isolation of the h u m a n sequences responsible for Wilms tumor [21]. Somatic cell hybrids have not only been informative in d e t e r m i n i n g whether the presence or absence of a specific c h r o m o s o m e can be associated with a certain type or stage of malignancy, they have been very helpful in targeting defined c h r o m o s o m e regions responsible for the altered transcription of a protein product. The altered p r o d u c t i o n of myc protein products has been found only in those hybrids in w h i c h the myc oncogene is reoriented near i m m u n o g l o b u l i n sequences [18, 19]. Correlations such as these are valuable diagnostic tools to distinguish patients with very defined clinical parameters of a specific type or stage of cancer.
Mapping Genes Associated with Cancer by In Situ Hybridization M a p p i n g and ordering cancer-associated genes by somatic cell h y b r i d i z a t i o n alone d e p e n d s on the ability to generate a sufficient n u m b e r of clones that retain different segments of the same c h r o m o s o m e or set of chromosomes. Because of the extensive c h r o m o s o m e instability and constant rearrangement between the h u m a n and rodent
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elements, this is often extremely difficult to accomplish. In addition, such hybrid panels often require constant monitoring and screening to ensure they contain the h u m a n elements that were selected for initially. Chromosomal in situ h y b r i d i z a t i o n localizes a DNA sequence directly to a precisely defined c h r o m o s o m e segment or band. The h y b r i d i z a t i o n signal is detected either with radioactively labeled (usually with :~H) DNA probes or probes labeled with nonradioactive c o m p o u n d s such as biotin 11-dUTP. The hybridization signal from nonradioactive labeled probes can then be detected with a variety of immunofluorescent c o m p o u n d s such as fluorescein (FITC) conjugated with avidin. In situ h y b r i d i z a t i o n has been used in conjunction with somatic cell h y b r i d i z a t i o n to regionally localize genes less than 0.5 kb to a precisely defined c h r o m o s o m e locus. 'The in situ technique permits visualization of genetic alterations associated with cancer at a m u c h higher level of resolution than cytogenetics or somatic cell hybridization. A series of probes that map to a c h r o m o s o m e region involved in a variety of malignancies will result in the construction of a physical map in w h i c h the order of genes can be ascertained in relation to the altered chromosome segment. For instance, using several probes derived from the l a m b d a light chain i m m u n o g l o b u l i n locus at 22q11, it was possible to determine the location of genes potentially responsible for the manifestation of several constitutional and malignant disorders in w h i c h c h r o m o s o m e 22 participated in a chromosome translocation event. These i n c l u d e d defining the breakpoint region of a 4:22 translocation observed in a patient with DiGeorge s y n d r o m e [4]: defining the breakpoint of the 9:22 translocation diagnostic of chronic myelogenous leukemia (CML) [10, 17]; defining the breakpoint of the 8;22 translocation characteristic of patients with variant Burkitt l y m p h o m a [16]; defining the sequences involved in a patient diagnosed with acute l y m p h o c y t i c leukemia (ALL) with a 9;22 rearrangement [5, 10]. In each case, the l a m b d a gene h e l p e d to define the order of translocation breakpoints in relation to each other and also h e l p e d to target specific sequences along c h r o m o s o m e 22 as potential sites that may contain cancer-associated loci. Four different probes that define breakpoint cluster region (bcr) loci have been m a p p e d in relation to each other as well as to the cancer breakpoint of both ALL and CML [10], using both in situ h y b r i d i z a t i o n and somatic cell hybridization. In some cases, in situ hybridization, especially with radioactively labeled probes, will detect alterations of a gene that cannot be defined cytogenetically. For example, in the ML-3 cell line established from a patient with acute myelogenous leukemia, there was no visible rearrangement of sequences from chromosome 22 to c h r o m o s o m e 17. However, in situ h y b r i d i z a t i o n with a c-sis oncogene probe that maps to the 22q13 region detected a reorientation of c h r o m o s o m e 22 sequences to the long arm of c h r o m o s o m e 17 [58]. In addition, it is possible to use in situ h y b r i d i z a t i o n to quantitate the presence or absence of sequences along a chromosome, w h i c h in some malignancies is manifested either as d u p l i c a t i o n s or deletions of a c h r o m o s o m e locus. Numerous other examples can be cited. In situ hybridization, therefore, has become an essential tool to define sequences or genes involved in a malignancy, and is being used to construct a physical map of genes along the c h r o m o s o m e in relation to other k n o w n DNA markers and cancer breakpoint regions. Maps generated in this way for c h r o m o s o m e regions consistently involved in neoplasia have increased the resolution of these areas of the h u m a n genome previously unattainable by cytogenetic evaluation alone. The m a p p i n g potential of in situ h y b r i d i z a t i o n technology has e x p a n d e d considerably as a result of the increased use of non-radioactive immunofluorescent c o m p o u n d s such as FITC conjugated to avidin [31-34, 44, 45, 55]. Recent i m p r o v e m e n t s in this technique now permit detection of signal from unique DNA probes that range in size from 0.2-0.5 kb in length as a result of repeated amplification with FITC [34]. The use of immunofluorescent c o m p o u n d s has significant advantages, especially for clinical
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diagnosis of malignant disorders where it is not always possible to generate analyzable metaphases. Unlike the radioactive in situ c o m p o u n d s , it is not necessary to expose t h e m for e x t e n d e d periods over an autoradiographic e m u l s i o n [31-34, 44, 451. In most cases, the preparations are m u c h cleaner with significantly less background than radioactive preparations. This enhances the overall quality and facilitates quantitation of signal over specific c h r o m o s o m e areas. One advantage that the radioactive detection m e t h o d still retains, however, is its ability to detect signal from very small DNA fragments less than 500 bp. A significantly larger quantity of DNA is necessary for detection of such small unique sequences with the non-radioactive method, and requires repeated amplifications with FITC to c o m p e n s a t e for this difficulty. In laboratories performing diagnostic evaluation of tumor-derived samples, it is important to maintain flexibility in establishing the c h r o m o s o m a l in situ h y b r i d i z a t i o n procedures, so that it is possible to switch from one to the other d e p e n d i n g on the tumor s a m p l e and DNA probe being used to evaluate a chromosomal alteration. Non-radioactive detection also has the ability to detect numerical and structural c h r o m o s o m e alterations in the interphase stage as well as in the m e t a p h a s e stage. In some tumors, such as b l a d d e r cancer, it is difficult to establish biopsy samples in vitro for e x t e n d e d periods and results in an insufficient number of analyzable metaphases (Sandberg, personal communication). Detection of c h r o m o s o m e abnormalities in interphase can be achieved in several ways. The most c o m m o n method involves the use of a chromosome-specific flow-sorted library [31,44, 45]. This results in " c h r o m o s o m e painting" and has been used to detect numerical aberrations such as trisomy 21 [44, 45], and a 4',11 translocation in a leukemia cell line [45]. In metaphases, the use of total h u m a n genomic DNA labeled with biotin has facilitated rapid screening of h u m a n - r o d e n t hybrid panels. In such hybrids, it is possible to detect as little as 1 × 107 bp of h u m a n DNA inserted into a rodent c h r o m o s o m e [15, 45]. More recent a p p l i c a t i o n s of fluorescent in situ h y b r i d i z a t i o n include detecting signal from m u l t i p l e probes either in metaphase or in the interphase cell [28-30, 55, 59]. In one case, it was possible to detect numerical and structural alterations of c h r o m o s o m e 1 in the leukemia cell line K562 using a combination of telomeric and centromeric probes each labeled with biotin and h y b r i d i z e d at differing concentrations [59]. In addition, studies with m u l t i p l e fluorophores now permit localization of several probes w i t h i n the same metaphase or interphase [28, 29, 55,561. This is of pivotal i m p o r t a n c e in clinical diagnosis, where both the linear relationship and organization of genes can be readily defined with probes that fluoresce different colors due to their activation at differing ultraviolet wavelengths. For example, in patients diagnosed with Ph + CML, it is possible to detect the 9q + c h r o m o s o m e by dual hybridization with bcr and abl probes each labeled with a different fluorophore [55]. W h e n counterstained, the fusion of the bcr-abl sequences was discerned as two distinctly colored fluorescent signals along the same c h r o m o s o m e [55]. In addition, m u l t i p l e copies of the translocation chromosome could be detected in the interphase stage. It is now possible to visualize fluorescent signals over chromosomes b a n d e d with c o m p o u n d s like DAPI and Hoechst 33258, w h i c h will elicit bands similar to G- or Rbands, respectively [6, 20]. The origin of c h r o m o s o m e s involved in cancer-associated numerical and structural abnormalities can thus be more precisely identified. The c o m b i n e d use of the radioactive and fluorescent in situ h y b r i d i z a t i o n method will provide an effective strategy to define numerical and structural alterations in all malignant types and stages.
Isolation of Sequences Consistently Altered in Neoplasia by Chromosome Microdissection Microdissection of h u m a n c h r o m o s o m e s is a relatively new and powerful tool in w h i c h DNA " m i c r o c l o n e s " are directly dissected from a c h r o m o s o m e region by m i c r o m a n i p u l a t i o n . The first microclones were derived from the m i c r o d i s s e c t i o n ot
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salivary gland polytene chromosomes of Diptera [53]. Such clones were isolated without any special treatment to distinguish a specific chromosome or band region. The chromosomes of interest, especially translocations, were thus identified on the basis of morphology alone [48]. More recently, microdissection is performed on fixed high-resolution banded chromosomes [3, 27, 35, 38, 52, 61]. DNA is dissected from a chromosome band or subregion that is precisely defined and thus the creation of DNA libraries from an identifiable target sequence along the chromosome is facilitated. The microdissection technique involves moving a fine needle, usually 0.1-0.5/xm in diameter, with a micromanipulator. DNA fragments are dissected from varying n u m b e r s of metaphases, from a few as 6 to as many as 50. The dissected fragments are pooled together and digested with a restriction enzyme, such as Rsal, which yields very small fragments. These fragments range in size from 50-350 bp [351 and when cut with other restriction enzymes, fragments up to 11 kbp can be generated {27]. The advantage of isolating such small DNA fragments is that the majority of them, about 80%, will be u n i q u e sequence clones without linked repetitive sequences [35l. This is a significant benefit, especially in isolating regions that most likely contain actively transcribed genes and disease-associated loci. These fragments can be inserted into an appropriate vector system and then amplified by the polymerase chain reaction (PCR) for further molecular characterization. The use of microdissection to isolate the genes involved in chromosome alterations in leukemia and solid tumors will provide a rapid means of creating DNA libraries from these altered regions. Libraries have already been generated from patients with neuroblastoma who manifest consistent alterations of the short arm of chromosome 1 [38]. The DNA clones isolated by chromosome microdissection can then serve as genetic markers or detect restriction fragment-length polymorphisms (RFLPs) that will identify at-risk or carrier individuals. Similar strategies have been undertaken to isolate clones from within the 11p13 chromosome region that contains the genes responsible for manifestation of the aniridia Wilms' tumor complex [14]. Multiple clones can be isolated and used to construct physical maps of these cancer-associated regions of the h u m a n genome. The u n i q u e sequence clones generated from microdissection are used as start points to pull out adjacent sequences along the chromosome, similar to the more conventional approach of walking along the chromosome. Once the precise location of each clone is confirmed either by in situ hybridization or somatic cell hybridization, they can be sequenced and compared to homologous segments from i n d i v i d u a l s who do not manifest any disease. Characterization ot cancer-associated altered DNA in this way will be essential at differing stages of the disease progression to help define the association of a specific stage of malignant progression with the manifestation of a cytogenetic or molecular abnormality. Ultimately, this fine dissection of the h u m a n genome will provide the necessary diagnostic markers to finally discern specific tumor types and stages of malignant progression. In summary, a n u m b e r of tools are now available to map cancer-associated genes. Used in combination with each other, they provide very powerful means to dissect vulnerable areas of the h u m a n genome that are responsible for the manifestation and progression of neoplasia. REFERENCES
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