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Definitions

• the invention relates to the area of cancer diagnostics and monitoring. More particularly it relates to the diagnosis and monitor ⁇ ing of cancers associated with chromosomal translocations t(l;14)(p32;qll). BACKGROUND OF THE INVENTION
• a method of diagnosing or assessing a neoplasm in a human comprising isolating DNA of a human from cells suspected of being neoplastic; determining whether a TCL-5 gene on said DNA has been rearranged, a rearranged TCL-5 gene indicating a neoplasm.
• a method of diagnos ⁇ ing or assessing a neoplasm in a human comprising isolat ⁇ ing from the human test cells suspected of being neoplastic; deter ⁇ mining quantity of expression products of TCL-5 genes in said test cells; comparing the quantity of TCL-5 gene expression products in said test cells with the quantity in control cells, said control cells isolated from healthy cells of the human or from a healthy human, an altered quantity of TCL-5 gene expression products in said test cells relative to said control cells indicating a neoplasm.
• a nucleic acid probe which is derived from chromosome 1 sequences located between a translocation junction of a lp + chromosome of a t(l;14)(p32;qll) translocation and a restriction enzyme site, wherein there are no other sites for said enzyme between said site and the translocation junction.
• a pair of nucleic acid primers are provided for amplifying translocation junctions of a lp + chromosome of a t(l;14)(p32;qll) translocation wherein one primer of the pair is derived from chromosome 1 sequences and one primer is derived from chromosome 14 sequences.
• the present invention thus provides the art with methods, probes and primers for diagnosis and monitoring certain neoplasms which render such diagnosis and monitoring routine and inexpensive.
• Figure 1 shows a representative trypsin-Giemsa banded karyotype of the 730-3 cell line: 47, XY, +17, t(l;13)(p32;q34), t(l;l4)(p32;qll).
• the lp + and 14q ⁇ chromosomes resulting from the t(l;14) translocation involving TCL-5 are designated; the other abnor ⁇ malities are indicated by arrows.
• Figure 2 shows analysis of the t(l;14) chromosomal breakpoint on the lp + chromosome in leukemic cell lines.
• Panel A shows hybrid ⁇ ization of DNA from t(l;14) cell lines with the TCR and pJK3.OS probe. DNA (10 ug) was isolated from cells of placenta (lanes 1 and 4), 730-3 (lanes 2 and 5) and DU.528 (lanes 3 and 6), and was digested with BamHI (lanes 1-3) or Hindm (lanes 4-6).
• Panels B-D shows restriction maps of regions surrounding the lp + chromosomal breakpoint in DU.528.
• Panel B shows the normal configuration of the TCR delta locus on chromosome 14.
• Panel C shows the rearranged TCL-5 locus on the lp + chromosome.
• Panel D shows the complemen ⁇ tary germline TCL-5 locus on chromosome 1. Black bars indicate chromosome 14 DNA sequences; white bars indicate chromosome 1 sequences. Probes are shown as hatched boxes. Abbreviations used are: E, EcoRI; B, BamHI; H, Hindm.
• Figure 3 shows that sequences juxtaposed to the TCR delta locus in the translocation chromosome of t(l;14) map to chromosome 1.
• DNA (10 ug/lane) from the following sources was digested with an excess of restriction enzyme Hind in, electrophoresed, transferred to a nitrocellulose filter, and hybridized with the p528H5.1 probe (see figure 2): mouse LMTK " cell line (lane 1); M44C12S5 hybrid retaining the 14q + chromosome from the translocation t(8;14)(q24;q32) (lane 2); GL5 hybrid retaining chromosome 4, 13, 14, 18, 20, 21, X and partial chromosomes 17 and 22 (lane 3); 401AD5EF3 hybrid retaining partial 8 and 22 and chromosomes 19, 21, and X (lane 4); 52-63cl7-17 hybrid retaining the 14q + chromosome from the translocation t(14;X)(q32;ql3) (lane 5); GM7299 hybrid
• Figure 4 depicts the 14q ⁇ chromosomal reciprocal breakpoint.
• Panel A shows a Southern filter hybridized with the p528B0.7 probe (see Figure 2) containing a germline chromosome 1 insert encompass ⁇ ing the t(l;14) breakpoint.
• DNA (10 ug/lane) from mouse cell line (lane 1), human placenta (lane 2), DU.528 cell line (lane 3), hybrid 726C122 (lane 4) and hybrid 726cl24 (lane 5) was digested with EcoRI, electrophoresed, and transferred to a nitrocellulose filter.
• Panel B shows restriction maps of the chromosome 14 TCR delta locus (black bar) and the chromosome 1 TCL-5 locus (white bar) that recombined to form the 14q ⁇ chromosome of the t(l;14) translocation (black and white bar).
• E EcoRI
• B BamHI
• H Hindm.
• Figure 5 compares the nucleotide sequence at the t(l;14) translocation junctions and the corresponding germline sequences.
• Panel A shows the lp chromosome breakpoint sequence.
• Panel B shows the I4q ⁇ chromosomal reciprocal breakpoint sequence.
• Germline chromosome 14 D-delta ⁇ and D-delta2 sequences are shown as previously published (Takihra, et al., (1988) Proc. Natl. Acad. Sci. USA 85:6097-6101). Brackets indicate heptamer signal sequences of the D-delta segments. The sequences shaded in gray are thought to represent N regions.
• FIG. 6 shows the expression of a TCL-5 transcript in various T-cell lines and a B cell line.
• Samples (10 ug) of total cellular RNA from five cell lines were electrophoresed in 1% agarose containing 2.2 M formaldehyde and then transferred to nitrocellulose.
• Duplicate filters were prepared and hybridized with the TCL-5 528B0.7 probe (panel A) or with the human ribosomal pA probe (Erickson et al.,
• Figure 7 demonstrates the there is a rearrangement of the TCL-5 locus in WM8 melanoma DNA.
• 10 ug of GM607 DNA used as a germline control (lanes A) or WM8 DNA (lanes B and C) were digested with the enzymes indicated, fractionated and hybridized with the P528B4.4 probe.
• Figure 8 depicts two models of the t(l;14) translocation event. D deltai and D delta2 segments are shown by the stippled and filled boxes, respectively. Chromosome 1 and 14 are shown by a single and a double line, respectively. In Panel A the translocation and D delta ⁇ -D delta2 attempted joining occur simultaneously. In Panel B the D deltai-D delta2 joining precedes the translocation. The trian ⁇ gle represents the breakpoint site on chromosome 1. DETAILED DESCRIPTION
• nucleic acid sequences of the TCL-5 oncogene which are provided by the present invention, neoplasms can be diagnosed, assessed or monitored.
• nucleic acid probes comprising portions of the TCL-5 gene can be used to hybridize to DNA isolated from cells suspected of being neoplastic.
• the DNA can be digested with restriction endonucleases to form discrete fragments. Detection of hybridized fragments in the isolated DNA of different sizes than those detected by the probe with control DNA samples indicates a TCL-5 rearrangement and neoplasia.
• the rearrangement may be associated with a t(l;14)(p32;qll) translocation, although other rear ⁇ rangements are possible which would be detected by the methods of the present invention, such as deletions, insertions and inversions.
• neoplasms generally provides a positive deter ⁇ mination that a malignant state exists. Further, by determining that a TCL-5 rearrangement has occurred a classification of the neoplasm can be made to a class of neoplasms involving such rearrangements. The members of a class of neoplasms may respond similarly to thera ⁇ pies, which is useful for choosing a course of treatment.
• a neoplasm may be assessed or monitored using the methods of the present invention. This generally provides a quantitative mea ⁇ sure of the amount of neoplastic cells remaining in a patient.
• the method of the present invention can be used to monitor the success of such therapy. This may involve quantitating the amount of rearranged TCL-5 genes detectable as compared to before therapy. Alternatively, this could involve quanti ⁇ tating the amount and type of TCL-5 gene products and comparing these with levels before therapy.
• TCL-5 rearrangements are determined in a human whose neoplasm has been in remission.
• TCL-5 rearrangements are detected, they provide a measure or indication of minimal residual disease, i.e. the recurrence of detectable neoplastic cells.
• An indication of mini ⁇ mal residual disease provides the clinician with notice to resume anti-neoplastic therapy.
• DNA of a human is iso ⁇ lated from cells which are suspected of being neoplastic.
• a sus ⁇ picion may be based on morphological, cytological, or other means relied on by clinicians. It is then determined whether the DNA iso ⁇ lated from the cells contains rearranged TCL-5 genes. While rear ⁇ rangements involving t(l;14)(p32;qll) translocations are specifically contemplated, other rearrangements involving TCL-5 could occur, such as insertions, deletions or inversions, which would lead to the neoplastic state.
• TCL-5 rearrangements have been found to occur in both leuke ⁇ mia and melanoma cells. However, it is not beyond the scope of the invention that other neoplasms would be associated with TCL-5 rear ⁇ rangements.
• neuroblastomas may be associated with TCL-5 rearrangements, as chromosome 1 band p32 has been shown to be rearranged in neuroblastoma cells. It is possible that a TCL-5 rearrangement is one of a number of genetic changes that occur dur ⁇ ing, and contribute to the oncogenic process. If the human is sus ⁇ pected of having leukemia, then T-lymphocytes are the preferred cell source for isolating DNA.
• melanocytes are the preferred cell source for isolating DNA.
• TCL-5 rearrange ⁇ ment may be inherited, in which case all somatic cells would carry the rearrangement. People carrying such inherited rearrangements may be prone to develop neoplasms.
• a TCL-5 rearrangement is determined by hybridizing a nucleic acid probe derived from chromosome 1 sequences.
• the probe sequences are those retained on the lp + chromosome after a t(l;l4)(p32;qll) translocation.
• the sequences are close enough to the translocation junction (where chromosome 1 sequences are joined to chromosome 14 sequences) that they lie between the translocation junction and a first restriction enzyme site; there are no other sites for the restric ⁇ tion enzyme between the translocation junction and the first site.
• Such probes hybridize to the TCL-5 RNA transcripts (expression products) and thus can be used to detect and quantify transcripts.
• Suitable probes are shown in Figure 2 and include p528B4.4, p528H5.1 and p528B0.7. Probes p528B4.4, p528H5.1 and p528B0.7, have been deposited at the American Type Culture Collection (ATCC) in
• Preferred probes are those whose sequences lie between the translocation junction and the first EcoRI site. These include those probes named above.
• the step of deter ⁇ mining is performed by amplification of particular sequences by the polymerase chain reaction technique, as is well known in the art. See, Mullis, Science, (1988), vol. 239, pp. 487-491; Scharf, Science, (1988), VQI. 233, pp. 1076-1078.
• the sequences preferably amplified are those which include the translocation junction of a lp + chromo ⁇ some resulting from a t(l;14)(p32;qll) chromosome.
• the primers for such amplification are single stranded nucleic acids, one of which comprises chromosome 14 sequences and one of which com ⁇ prises chromosome 1 sequences.
• the chromosome 14 sequences are prefera ⁇ bly derived from the TCR-delta locus encoding the T cell antigen receptor. Most preferably these comprise sequences derived from D-delta-2 or J-delta-l segments of the locus.
• a suitable probe of this kind has been deposited at the ATCC as accession no. 67996, and is known as pl01J-delta-E5.0.
• Preferred chromosome 1 primers are derived from sequences on probe p528B0.7, which spans the translocation breakpoint on chromosome 1.
• control cells are isolated from healthy cells of the same human supplying the test cells or from a healthy human. Plaeental cells may also be used.
• Methods of quantifying protein and nucleic acids are well known in the art. For example, radiolabeled nucleic acid probes can be used and the amount hybridizing can be determined by autoradiography and densitometry. Other means of quantifying may also be used.
• Gene products which have been detected in cells carry ⁇ ing t(l;14)(p32;qll) translocations are about 5.4 kb, about 2.2 kb, and about 2.6 kb RNA transcripts.
• the protein products of these transcripts can be detected by antibodies which recognize epitopes on the proteins.
• Antibodies recognizing epitopes unique to specific protein products can be used to distinguish the populations of TCL-5 proteins.
• Meth ⁇ ods for quantitating protein products are known in the art and may be used as are convenient.
• One convenient method is the ELISA tech ⁇ nique, which employs a colorimetric assay wherein the color corre ⁇ lates with the amount of antigen present. Means of obtaining anti ⁇ bodies for specific protein products are also routine and well known in the art.
• Nucleic acid probes and primers discussed above which can be used in the practice of the method of the present invention are also provided. They can be isolated from biological sources such as the deposited cell lines or plasmids mentioned above, or they can be syn ⁇ thesized according to known sequences, for example those shown in Figure 5. Methods for preparing and labeling probes are well known in the art. Methods for preparing primers are also well known.
• This example describes the identification of the t(l;14)(p32;qll) chromosomal breakpoint in leukemic cells.
• the t(l;l4) chromosomal translocation has been described in the leukemic cells of a 16 year-old boy with acute T lymphoblastic leukemia (Hershfield et al., (1984) Proc. Natl. Acad. Sci. USA 81:253-257).
• Two cell lines, DU.528 (Kurtzberg et al., (1985) J. Exp. Med. 162:1561-1578) and 730-3 (Kurtzberg, unpublished results), derived from the leukemic cells prior to and after chemotherapy, respectively, showed an apparently identical karyotype.
• TCR T-cell receptor
• This probe which con ⁇ tains a 3.0 kb Sstl fragment located within the TCR delta locus as illustrated in Figure 2B, hybridizes to a 10.5 kb BamHI rearranged fragment in both DU.528 and 730-3 Bam Hi-digested DNAs (Fig. 2A, lanes 2,3). Since the pJK3.0S insert has an internal Hindm site, the probe detects two germline restriction fragments of 3.4 kb and 6.2 kb in Hindm-digested plaeental DNA (Fig. 2A, lane 4).
• pJK3.0S detects a 3.4 kb germline restriction fragment and a 9.8 kb rearranged restriction fragment in Hindm-digested DNAs of DU.528 and 730-3 cells (Fig. 2 A).
• t(l;14) breakpoint probably occurred within the 9.8 kb Hindm fragment detected by the pJK3.0S probe (Fig. 2A).
• the chromosome 14 p528E5.0 probe containing a 5.0 kb EcoRI fragment as illustrated in Fig. 2B, was used to isolate and map the complementary germline TCR delta locus from a human plaeental library (Fig. 2B).
• a repeat-free 5.1 kb Hindm fragment located close to the breakpoint, where the restriction enzyme sites had diverged from the normal delta locus restriction sites (Fig. 2C) was cloned into pUC19 (p528H5.1) to be used in determining its chro ⁇ mosomal origin.
• Probe p528H5.1 was hybridized to a Southern filter with bound DNA from rodent x human hybrid cells containing either human chromosome l or human chromosome 14. The presence in the hybrids of the 5.1 kb Hindm fragment correlates only with the pres ⁇ ence of human chromosome 1 (Fig. 3), demonstrating that we have cloned the t(l;14) breakpoint on the DU.528 lp + chromosome (lqter-) lp32::14qll-> 14qter).
• the p528H5.1 probe was also used to screen a human plaeental library in order to clone the normal chro ⁇ mosome 1 counterpart of the breakpoint region (Fig. 2D).
• This example demonstrates the isolation of the reciprocal translocation joining segment.
• the p528B0.7 probe contains a germline 0.7 kb BamHI insert from chromosome 1 that includes the DU.528 lp + translocation breakpoint (as illustrated in Fig. 2D).
• an EcoRI digest of DU.528 DNA showed a germline fragment of 17.5 kb and rearranged fragments of 8 kb and 14 kb (Fig. 4A, lane 3).
• the 17.5 kb EcoRI fragment corresponds to the germline chromosome 1 fragment detected in both the DU.528 and human control DNAs.
• the 14 kb EcoRI rear ⁇ ranged fragment corresponds to the EcoRI segment encompassing the breakpoint on the DU.528 chromosome lp + , a con ⁇ clusion based on restriction analysis using the p528EB2.7 probe.
• the p528EB2.7 probe spans the DU.528 chromosome lp + breakpoint, as depicted in Fig. 2C, and detects the same size 14 kb rearranged frag ⁇ ment in DU.528 EcoRI-digested DNA.
• the 8 kb EcoRI rearranged fragment (Fig. 4A, lane 3) could correspond to the translocation junction on the derivative chromo ⁇ some 14q ⁇ in DU.528.
• phage clones that hybridized to the p528B0.7 probe but not to the p528EB2.7 probe (a probe that hybridizes to DU.528 phage clones with inserts covering the chromosome lp + junction or the nor ⁇ mal chromosome 1 counterpart) were isolated and characterized in detail.
• hybrid lines 726cl22 and 726cl24 which retained the 8 kb EcoRI rearranged fragment corresponding to the 14q ⁇ (14pter ⁇ 14qll::lp32-> lpter) chromosomal junction region (Fig. 4A, lanes 4,5).
• the two hybrids also retained the L-MYC locus which has been mapped to chromosome region lp32 (Nau et al., 1985, Nature 318:69-73) but did not retain the nerve growth factor beta locus assigned to chromosome region lp22 or lpl3 (Munke et al., 1984, Somat. Cell. Genet.
• This example shows that the chromosome 14 sequences involved in the t(l;14)(p32;qll) translocation are in the D-delta locus.
• the nucleotide sequence of the translocation junction is also shown.
• DNA frag ⁇ ments were subcloned in M13mpl8 or M13mpl9 and their sequences were determined by using the Sanger dideoxy method (Sanger et al., 1977. Proc. Natl. Acad. Sci USA 74:5463-5467) with Sequenase (United States Biochemical, Cleveland). The nucleotide sequences of the translocation junction sites and the normal counterparts are presented in Fig. 5.
• the chromosome 1 sequences at the junction on the lp + chromosome and the 14q ⁇ chromosome are continuous in relation to the germline chromosome 1, thus demon ⁇ strating the reciprocity of the translocation in relation to chromo ⁇ some 1.
• Examination of the sequence from chromosome 14 at the translocation junction identified features which may have influenced the translocation process.
• the joining on the lp + chromosome occurred at the D-delta2 segment (Fig. 5A).
• D-delta2 segment normally possesses the 3' and 5' heptamer and nonamer recombination signal sequences used during D segment rear ⁇ rangements (Takihara et al., 1988, Proc. Natl. Acad. Sci. USA 85:6097-6101). Strikingly, the t(l;14) breakpoint on the lp + chromo ⁇ some occurred in D-delta2 precisely at a site where normal D seg ⁇ ment rearrangement occurs, i.e., at the site immediately 3' to the 5' signal sequences, such that these sequences are lost during the recombination process (Sakano et al., 1981, Nature 290:562-565).
• the chromosome 14 sequences from the reciprocal chromo ⁇ some 14q ⁇ side of the breakpoint demonstrate that the junction site is at the D-deltai segment of the TCR delta locus (Fig. 5B). Analogous to normal D segment rearrangement (Sakano et al., 1981, Nature 290:562-565), the reciprocal breakpoint has occurred precisely 5 1 to the 3' D-deltai signal sequences. Extra nucleotides present at both the junction sites on chromosomes lp + and 14q ⁇ are highlighted by the stippled boxes in Fig. 5 and are not derived from the normal germline sequences. Such additional nucleotides at breakpoints in B- and T-cell translocations may represent N regions (Tsujimoto et al.,
• This example demonstrates that there is a gene located at the translocation junction on chromosome 1.
• chromosomal loci aberrantly juxtaposed to immunoglobulin super family loci by chromosomal rearrangements are inappropriately transcriptionally active, a feature which is thought to reflect the transcription of a gene contributing directly to the malignant process (reviewed in Finger et al., 1989, "Involvement of the Immunoglobulin Loci in B-cell Neoplasms, in Immunoglobulin Genes (F. Alt, T. Honjo and T.H. Rabbitts, eds.) Academic Press, Ltd. London, pp. 221-230).
• Total cellular RNAs used for Northern blot analyses were from 730-3, a T-cell ALL cell line (TALL-101), a gamma/delta-producing T-cell line (Peer), an alpha/beta-producing T-cell line (Jurkat), and a pre-B-cell leukemia cell line (697).
• Hybridization of a filter retaining these RNAs to a ribosomal probe indicates that there are similar amounts of 28S ribosomal RNA in sample lanes (Figure 6B).
• the same RNA preparations on a duplicate filter show varying sizes and amounts of transcripts hybridizing to the chromosome 1 probe, 528B0.7 ( Figure 6A).
• the 528B0.7 probe detects a 5.4 kb RNA tran ⁇ script and possibly a low level of 2.2 kb and 2.6 kb RNA transcripts in the T-cell lines Jurkat and TALL-101.
• the gene represented by these transcripts, located at the translocation junction on chromosome 1, is designated TCL-5 (T-cell leukemia/lymphoma-5).
• TCL-5 T-cell leukemia/lymphoma-5
• a barely detectable level of a 5.4 kb TCL-5 RNA transcript in the 730-3 and Peer T-cell lines and the 697 pre-B cell line may be due to either low level expression of TCL-5 or to nonspecific cross-hybridization to 28S ribosomal RNA.
• the 528B0.7 probe detects a high level of expression of the 2.2 kb and 2.6 kb TCL-5 RNA transcripts, suggesting that an alteration of TCL-5 gene expression in 730-3 cells is directly involved in the neoplastic process in this human T-cell leukemia.
• This example demonstrates that the TCL-5 locus is rearranged in a melanoma cell line with a lp32 chromosomal abnormality.
• CMM human cutaneous malignant melanomas
• the p528B4.4 probe which contains a 4.4 BamHI fragment derived from ehromosome 1 adjacent to the breakpoint on the lp + chromosome as illustrated in Fig. 2D, detected a genomic rearrangement in DNA from the primary melanoma cell line WM8.
• Two DNA preparations from the WM8 cell line yielded identical results (Fig. 7, lanes B,C).
• the WM8 cell line carries a del(l)(p32) and numerous other chromo ⁇ somal aberrations (M. Herlyn and P.C. Nowell, unpublished results).
• Genomic rearrangements detected in WM8 DNA compared to human control DNA using several different restriction enzymes allowed us to exclude restriction fragment length polymorphisms as an explanation of the new rearranged fragments.
• the lower intensity of the rear ⁇ ranged restriction fragments relative to the germline restriction fragments may be due to a low copy number of the del(l)(p32) chromosome relative to the other chromosomes 1 observed in WM8, which is a hypotetraploid cell line.

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