WO1997038083A2 - New member of the multiple-tumor aberrant growth gene family - Google Patents

Abstract

The present invention relates to a new member of the multi-tumor Aberrant Growth (MAG) gene family located on the short arm of chromosome 3 and involved in a variety of benign and malignant tumors. The gene and its derivatives may be used in various diagnostic and therapeutic applications.

Description

NEW MEMBER OF THE MULTIPLE-TUMOR ABERRANT GROWTH GENE FAMILY

The present invention relates to the identification of a new translocation or fusion partner of members of the multi-tumor aberrant growth (MAG) gene family, the members of which are frequently associated with aberrant cell growth as found in a variety of both benign and malignant tumors.

The invention in particular relates to the identification of the PSAFP-1 gene as the broadly acting HMG (High Mobility Group) fusion partner involved in a number of tumors, including but not limited to the mesenchymal tumors hamartomas (e.g. breast and lung), lipo as, pleomorphic salivary gland adenomas, uterine leiomyomas, angio yxomas, fibroadenomas of the breast, polyps of the endometrium, atherosclerotic plaques, and other benign tumors as well as various malignant tumors, including but not limited to sarcomas (e.g. rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid) . The invention relates in particular to the use of the PSAFP-1 gene and its derivatives in diagnosis and therapy.

With respect to diagnosis, truncation/inactivation of PSAFP-1 by various mechansims (point mutation, deletion, chromosome translocation, etc.) and detected at the level of DNA, mRNA or protein is claimed. With respect to therapy, restauration of the genetic defect is claimed (gene therapy using gene targeting and gene transfer technologies) .

Multiple independent cytogenetic studies have firmly implicated region ql3-ql5 of chromosome 12 in a variety of benign and malignant solid tumor types. Among benign solid tumors, involvement of 12ql3-ql5 is frequently observed in benign adipose tissue tumors [1] , uterine leiomyomas [2, 3] , and pleomorphic adenomas of the salivary glands [4, 5] . Involvement of the same region has also been reported for endometrial polyps [6, 7] for he angio- pericytoma [8] , and for chondromatous tumors [9, 10, 11, 12] . Recently, the involvement of chromosome 12ql3-ql5 was reported in pulmonary chondroid hamartoma [13, 14] . Finally, several case reports of solid tumors with involvement of chromosome region 12ql3-ql5 have been published; e.g. tumors of the breast [15, 16] , diffuse astrocytomas [17] , and a giant-cell tumor of the bone [18] . Malignant tumor types with recurrent aberrations in 12ql3-ql5 include myxoid liposarcoma [19], soft tissue clear-cell sarcoma [20, 21, 22] , and a subgroup of rhabdomyosarcoma [23] .

Although these studies indicated that the same cytogenetic region of chromosome 12 is often involved in chromosome aberrations, like translocations, in these solid tumors, the precise nature of the chromosome 12 breakpoints in the various tumors is still not known. Neither was it established which genes are affected directly by the translocations . In previous physical mapping studies [39] , the chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, and uterine leiomyo a were mapped between locus D12S8 and the CHOP gene and it was shown that D12S8 is located distal to CHOP. Recently, it was also found by FISH analysis that the chromosome 12q breakpoints in a hamartoma of the breast, an angiomyxoma and multiple pulmonary chondroid hamartomas are mapping within this DNA interval. In an effort to molecularly clone the genes affected by the chromosome 12ql3-ql5 aberrations in the various tumors, the present inventors chose directional chromosome walking as a structural approach to define the DNA region encompassing these breakpoints.

As a starting point for chromosome walking, locus D12S8 was used. During these walking studies, it was shown that the chromosomal breakpoints as present in a number of uterine leio yoma-derived cell lines are clustered within a 445 kb chromosomal segment which has been designated Uterine Leiomyoma Cluster Region on chromosome ____ (ULCR12) [24] . Subsequently, it was found that a 1.7 Mb region on chromosome 12 encompasses the chromosome 12 breakpoints of uterine leiomyoma-, lipoma-, and salivary gland adenoma- cells, with the breakpoint cluster regions of the various tumor types overlapping [25] . This 1.7 Mb region on the long arm of chromosome 12, which contains ULCR12 obviously, was designated Multiple Aberration Region (MAR) to reflect this feature. In a regional fine mapping study, MAR was recently assigned to 12ql5. It has thus been found that essentially all breakpoints of chromosome 12 map in a 1 7 Mb region indicated herein as the "Multiple Aberration Region" or MAR Further research revealed that m this region a member of the High Mobility Group gene family, the HMGI-C gene, can be identified as a postulated multi-tumor aberrant growth gene (MAG)

Members of the MAG gene family are thus involved in all types of mutations that might lead to aberrant cell growth. One type of mutation are the translocations m which part of a MAG gene is exchanged with part of another gene. The part of the other gene is indicated as the "translocation or fusion partner" Translocation partners may be used in therapy and diagnosis, like the MAG genes.

According to the present invention it has now been found that a gene located at the short arm of chromosome 3 can act as the translocation partner of the HMGI-C gene. This gene is called "PSAFP-1".

The present invention now provides for this translocation partner gene or derivatives thereof in isolated form and its use in diagnostic and therapeutic applications .

In this application "MAG gene(s) " refers to genes associated with multiple aberrant cell growth. It has been found that the PSAFP-1 gene is not only the translocation partner of certain MAG genes but if affected by some kind of inactivating mutation may also induce aberrant growth. Therefore, by definition the term "MAG gene(s)" also includes the newly identified PSAFP-1 gene.

The term "wildtype cell" is used to indicate the cell not harbouring an aberrant chromosome or to a cell having a physiological expression level of the relevant gene. "Wildtype" or "normal" chromosome refers to a non- aberrant chromosome . The present invention provides for various diagnostic and therapeutic applications that are based on the information that may be derived from the gene. This information encompasses its nucleotide sequence or the amino acid sequence of the gene product derived from the gene.

The aberration in cell growth may be directly or indirectly caused by the physical breaks that occur in the gene or its vicinity, or by other types of mutations in the gene or its vicinity. On the other hand the aberration in cell growth may be caused by non-physiological expression forms of the gene. These non-physiological expression forms may be caused by the break, or may be due to stimulus that deactivates the gene. At present the exact mechanism or origin of the aberrant cell growth is not yet unraveled. However, exact knowledge on this mechanism is not necessary to define methods of diagnosis or treatment.

Diagnostic methods according to the invention are thus based on the fact that an aberration in a chromosome results in a detectable alteration in the chromosomes' appearance or biochemical behaviour. A translocation, for example will result in a first part of the chromosome (and consequently of the MAG gene) having been substituted for another (second) part (further referred to as "first and second substitution parts") . The first part will often appear someplace else on another chromosome from which the second part originates. As a consequence hybrids will be formed between the remaining parts of both (or in cases of triple translocations, even more) chromosomes and the substitution parts provided by their translocation partners. Since it has now been found that the breaks occur in a MAG gene this will result in hybrid gene products of that MAG gene. Markers, such as hybridising molecules like RNA, DNA or DNA/RNA hybrids, or antibodies will be able to detect such hybrids, both on the DNA level, and on the RNA or protein level.

For example, the transcript of a hybrid will still comprise the region provided by the remaining part of the gene/chromosome but will miss the region provided by the substitution part that has been translocated. In the case of inversions, deletions and insertions the gene may be equally afflicted.

Translocations are usually also cytogenetically detectable. The other aberrations are more difficult to find because they are often not visible on a cytogenetical level. The invention now provides possibilities for diagnosing all these types of chromosomal aberrations.

In translocations markers or probes based on the MAG gene for the remaining and substitution parts of a chromosome in situ detect the remaining part on the original chromosome but the substitution part on another, the translocation partner.

In the case of inversions for example, two probes will hybridise at a specific distance in the wildtype gene. This distance might however change due to an inversion. In situ such inversion may thus be visualized by labeling a set of suitable probes with the same or different detectable markers, such as fluorescent labels. Deletions and insertions may be detected in a similar manner.

According to the invention the above in situ applications can very advantageously be performed by using FISH techniques. The markers are e.g. two cosmids one of which comprises a first part of the PSAFP-1 gene, while the other comprises a second part. Both cosmids are labeled with different fluorescent markers, e.g. blue and yellow. The normal chromosome will show a combination of both labels, thus giving a green signal, while the translocation is visible as a blue signal on the remaining part of one chromosome (e.g. 12) while the yellow signal is found on another chromosome comprising the substitution part. In case the same labels are used for both probes, the intensity of the signal on the normal chromosome will be 100%, while the signal on the aberrant chromosomes is 50%. In the case of inversions one of the signals shifts from one place on the normal chromosome to another on the aberrant one.

In the above applications a reference must be included for comparison. Usually only one of the two chromosomes is afflicted. It will thus be very convenient to use the normal chromosome as an internal reference. Furthermore it is important to select one of the markers on the remaining or unchanging part of the chromosome and the other on the substitution or inverted part. As an alternative a combination of probes based on both translocation or fusion partners may be used.

Furthermore it was found that breaks might also occurr outside the gene, i.e. 5' or 3' thereof. The choice of the probes will then of course include at least one probe hybrising to a DNA sequence located 5' or 3 ' of the gene.

"Probes" as used herein should be widely interpreted and include but are not limited to linear DNA or RNA strands, Yeast Artificial Chromosomes (YACs) , or circular DNA forms, such as plasmids, phages, cosmids etc.. These in situ methods may be used on metaphase and interphase chromosomes .

Besides the above described in situ methods various diagnostic techniques may be performed on a more biochemical level, for example based on alterations in the DNA, RNA or protein, or on changes in the physiological expression level of the gene.

Basis for the methods that are based on alterations in the chromosome's biochemical behaviour is the fact that by choosing suitable probes, variations in the length or composition in the gene, transcript or protein may be detected on a gel or blot. Variations in length are visible because the normal gene, transcript (s) or protein(s) will appear in another place on the gel or blot then the aberrant one(s) . In case of a translocation more than the normal number of spots will appear.

Variations in the composition of the DNA, in particular in the PSAFP-1 gene and resulting in altered function or inactivation of the gene, mutation detection can be performed using standard technologies for mutation detection (see Laboratory Protocols for Mutation Detection. Ulf Landegren, Oxford University Press, 1996, ISBN 0 19 857795 8. (For example: PCR SSCP, SSCP and heteroduplex analysis, REF & ddF (restriction endonuclease and dideoxy fingerprinting) , DGGE, CDCE, LSSP-PCR, CCM, FAMA, EMC, MREC, SSO, PASA-PCR, solid-phase inisequencing, multiplex solid- phase fluorescent primer extension, OLA, LCR, UHG, DNA sequencing, FISH, PCR, RT-PCR, antibodies, PTT, MAMA, RED, Northern blotting, Southern blotting) .

Based on the above principle the invention may thus for example relate to a method of diagnosing cells having a non-physiological proliferative capacity, comprising the steps of taking a biopsy of the cells to be diagnosed, isolating a suitable PSAFP-1 gene-related macromolecule therefrom, and analysing the macromolecule thus obtained by comparison with a reference molecule originating from cells not showing a non-physiological proliferative capacity, preferably from the same individual. The PSAFP-1 gene-related macromolecule may thus be a DNA, an RNA or a protein.

In a specific embodiment of this type of diagnostic method the invention comprises the steps of taking a biopsy of the cells to be diagnosed, extracting total RNA thereof, preparing a first strand cDNA of the mRNA species in the total RNA extract or poly-A-selected fraction (s) thereof, which cDNA comprises a suitable tail; performing a PCR using a PSAFP-1 gene specific primer and a tail-specific primer in order to amplify PSAFP-1 gene specific cDNA's; separating the PCR products on a gel to obtain a pattern of bands; evaluating the presence of aberrant bands by comparison to wildtype bands, preferably originating from the same individual. As an alternative amplification may be performed by means of the Nucleic Acid Sequence-Based Amplification (NASBA) technique [81] or variations thereof.

In another embodiment the method comprises the steps of taking a biopsy of the cells to be diagnosed, isolating total protein therefrom, separating the total protein on a gel to obtain essentially individual bands, optionally transfering the bands to a Western blot, hybridising the bands thus obtained with antibodies directed against a part of the protein encoded by the remaining part of the PSAFP-1 gene and against a part of the protein encoded by the substitution part of the PSAFP-1 gene; visualising the antigen-antibody reactions and establishing the presence of aberrant bands by comparison with bands from wildtype proteins, preferably originating from the same individual .

In a further embodiment the method comprises taking a biopsy of the cells to be diagnosed; isolating total DNA therefrom; digesting the DNA with one or more so- called "rare cutter" restriction enzymes (typically "6- or more cutters") ; separating the digest thus prepared on a gel to obtain a separation pattern; optionally transfering the separation pattern to a Southern blot; hybridising the separation pattern in the gel or on the blot with a set of probes under hybridising conditions; visualising the hybridisations and establishing the presence of aberrant bands by comparison to wildtype bands, preferably originating from the same individual. Changes in the expression level of the gene may be detected by measuring mRNA levels or protein levels by means of a suitable probe.

Diagnostic methods based on abnormal expression levels of the gene may comprise the steps of taking a sample of the cells to be diagnosed; isolating mRNA therefrom; and establishing the presence and/or the (relative) quantity of mRNA transcribed from the MAG gene of interest in comparison to a control. Establishing the presence or (relative) quantity of the mRNA may be achieved by amplifying at least part of the mRNA of the MAG gene by means of RT-PCR or similar amplification techniques. In an alternative embodiment the expression level may be established by determination of the presence or the amount of the gene product (e.g. protein) by means of for example monoclonal antibodies.

The diagnostic methods of the invention may be used for diseases wherein cells having a non-physiological proliferative capacity are selected from the group consisting of benign tumors, such as the mesenchymal tumors hamartomas (e.g. breast and lung) , adipose tissue tumors (e.g. lipomas) , pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of the endometrium, atherosclerotic plaques, and other benign tumors as well as various malignant tumors, including but not limited to sarcomas (e.g. rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid) . The invention is not limited to the diagnosis and treatment of so-called benign and malignant solid tumors, but the principles thereof have been found to also apply to haematological malignancies like leukemias and lymphomas .

Recent publications indicate that atherosclerotic plaques also involve abnormal proliferation [26] of mainly smooth muscle cells and it was postulated that atherosclerotic plaques constitute benign tumors [27] . Therefore, this type of disorder is also to be understood as a possible indication for the use of the MAG gene family, in particular in diagnostic and therapeutic applications.

As already indicated above, it is sometimes found that in certain malignant tumors MAG gene function is abrogated. Until now the relevance of this observation is not understood. Another aspect of the invention thus relates to the implementation of the identification of the defective PSAFP-l gene in therapy. The invention for example provides functional molecules of the PSAFP-l gene for use in the treatment of diseases involving cells having a non- physiological proliferative capacity by correcting the gene defect. The defect may thus be repaired by means of introducing wildtype PSAFP-l DNA into non-physiologically proliferating cells which in turn may result in a normalisation of the cell growth.

As already indicated above it is sometimes found that in certain malignant tumors the expression level of these types of MAG genes is increased [28] . Until now the relevance of this observation was not understood. Another aspect of the invention thus relates to the implementation of the identification of the PSAFP-l gene in therapy. The invention for example provides anti-sense molecules or expression inhibitors of the PSAFP-l gene for use in the treatment of diseases involving cells having a non- physiological proliferative capacity by modulating the expression of the gene. A non-physiological high expression may thus be normalised by means of antisense RNA that is either administered to the cell or expressed thereby and binds to the mRNA, or antibodies directed to the gene product, which in turn may result in a normalisation of the cell growth.

The invention thus provides derivatives of the PSAFP-l gene and/or its immediate environment for use in diagnosis and the preparation of therapeutical compositions, wherein the derivatives are selected from the group consisting of sense and anti-sense cDNA or fragments thereof, transcripts of the gene or fragments thereof, antisense RNA, triple helix inducing molecule or other types of "transcription clamps", fragments of the gene or its complementary strand, proteins encoded by the gene or fragments thereof, protein nucleic acids (PNA) , antibodies directed to the gene, the cDNA, the transcript, the protein or the fragments thereof, as well as antibody fragments. Besides the use of direct derivatives of the genes and their surroundings (flanking sequences) in diagnosis and therapy, other molecules, like expression inhibitors or expression enhancers, may be used for therapeutic treatment according to the invention. An example of this type of molecule are ribozymes that destroy RNA molecules. Besides the above described therapeutic and diagnostic methods the principles of the invention may also be used for producing a transgenic animal model for testing pharmaceuticals for treatment of PSAFP-l gene related malignant or benign tumors and atherosclerotic plaques. The relevant sequence data are given in Fig. 1 in which:

A. shows the total cDNA of the PSAFP-l gene, a human fusion partner gene of MHGI-C. The cDNA was isolated through RACE product pCH223 from a pleomorphic salivary gland adenoma. The start and stop codons are underlined;

B. shows the derived amino acid sequence of the open reading frame; C. shows the HMGI-C part (top) and PSAFP-l part

(bottom) of the RACE product pCH223; and

D. shows the amino acid sequence of the PSAFP-l translocation part of the fusion product fused to the DNA binding domain 3 (DBD3) of HMGI-C. The YAC clones comprising the PSAFP-l gene are

CEPH mark 1 YACs 145F7, 275A12, and 405C8 (spanning about 760 kb) . Fig. 2 shows the long range physical map encompassing the PSAFP-l gene. The isolation of the gene and the claimed diagnostic tests and therapeutic methods may all be reduced to practice by the skilled person while using standard techniques as for example disclosed in Laboratory Protocol for Mutation Detection. Ulf Landegren, Oxford University Press, 1996, ISBN 0 19 857795 8. Examples of these techniques are: PCR SSCP, SSCP and heteroduplex analysis, REF & ddF (restriction endonuclease and dideoxy fingerprinting) , DGGE, CDCE, LSSP-PCR, CCM, FAMA, EMC, MREC, SSO, PASA-PCR, solid-phase minisequencing, multiplex solid- phase fluorescent primer extension, OLA, LCR, UHG, DNA sequencing, FISH, PCR, RT-PCR, antibodies, PTT, MAMA, RED, Northern blotting, Southern blotting.

EXAMPLE 1

Isolation of PSAFP-l gene

In order to study the possible role of translocation partner genes of HMGIC in benign solid tumors, we have analyzed pleomorphic adenoma of the parotid gland. 3 ' -RACE experiments were performed to establish whether or not HMGIC-derived fusion transcripts were expressed in these type of tumors. 3 ' -RACE was performed using the 3 ' -exon trapping protocol of GIBCO/BRL with slight modifications. For first strand cDNA synthesis, adapter primer (AP2; 94M2363) AAG GAT CCG TCG ACA TC{T)₁₇ was used. For both initial and secondary rounds of PCR, the universal amplification primer (UAP2; 95L736) CUA CUA CUA CUA AAG GAT CCG TCG ACA TC was used as 'reverse primer¹. In the first PCR round the following HMGIC-specific 'forward primers' were used: (i; 94N1501) 5' -CTT CAG CCC AGG GAC AAC-3 ' (exon 1) , (ii; 94N701) 5' -CAA GAG GCA GAC CTA GGA-3' (exon 3) , or (iii; 94M1892) 5 ' -AAC AAT GCA ACT TTT AAT TAC TG-3 ' O'-UTR) . In the second round the following HMGIC-specific forward primers (nested primers as compared to those used in the first round) were used: (i; 94N1502) 5'-CAU CAU CAU CAU CGC CTC AGA AGA GAG GAC-3 ' (exon 4) , or (iii; 94M1908) 5' -CAU CAU CAU CAU TTG ATC TGA TAA GCA AGA GTG GG-3 ' (3'-UTR) . CUA/CAU-tailing of the nested, specific primers allowed the use of the directional CloneAmp cloning system (GIBCO/BRL) . 3 ' -RACE analysis of total RNA resulted in the identification of a PCR product of 617 bp. This product was molecularly cloned into the TA-cloning kit vector and, subsequently, sequenced. Comparison of these data to the HMGIC sequences revealed ectopic sequences, which presumably correspond to a pleomorphic adenoma fusion partner gene of HMGIC (PSAFP-l) . These sequences appeared to be fused to HMGIC sequences with a sequence diversion point immediately down-stream of HMGIC exon 3, indicating that a rearrangement in the HMGIC gene had occurred within the large intron (intron 3) of the gene. This is in the same region where most of the translocation breakpoints have been found in a variety of benign tumors. The PSAFP-l sequences contained stop codons in all three possible reading frames. In phase with the HMGIC reading frame, a small open reading frame was present coding for 31 amino acids. There was also a putative poly-adenylation signal (ATTAAA) in the ectopic sequences, upstream of the site where the oligo-d(T) -primed cDNA synthesis started. This could indicate that the ectopic sequences correspond to a transcribed gene sequence (the putative PSAFP-l gene) . This was confirmed by Northern blot analysis. Commercially obtained multiple tissue Northern blots (Clontech) were screened with a probe specific for the ectopic sequences (pCH223) . A transcript of approximately 1.2 kb was detected in all adult human tissues tested, including brain, heart, kidney, liver, lung pancreas, placenta, and skeletal muscle. Furthermore, a transcript of approximately 5 kb was found exclusively in brain and pancreas.

To establish the chromosomal origin of the ectopic sequences, CASH analysis was performed using the NIGMS human/rodent somatic cell hybrid mapping panel 2 (Corieil Cell Repositories) . These studies revealed that they were derived from chromosome 3. In order to more precisely map these sequences, the CEPH mark 1 and 3 YAC libraries were screened with PCR probe pCH223, which corresponds to the ectopic sequences. This resulted in the isolation of three CEPH-A YAC clones (145A7, 275A12 and 405C8) and 13 mega-YAC clones (750F1, 752F4 , 752F5, 768A7, 768B7, 768D2, 775B3,

775B6, 805F12, 850A6, 894H3, 918A4 and 944D4) . A YAC query at http: //www-genome.wi .mit .edu/cgi-bin/cintig/phys_map showed that these mega-YACs all mapped within M.I.T YAC contig WC-3.10 located at 3pl4.2-3p21. A long range physical map was constructed with the overlapping YAC clones 145F7, 275A12, 405C8, and 850A6 and, subsequently, the relative position of the ectopic sequences (pCH223) within this map was established. FISH analysis of normal metaphase chromosomes using YAC clone 850A6 as molecular probe confirmed the localization of this YAC to 3pl4. cDNA clones corresponding to the chromosome 3-derived PSAFP-l sequences were isolated and the nucleotide sequence thereof established. A routine BLAST search by e-mail at blast@-ncbi.nlm.gov revealed that the PSAFP-l sequences fused to HMGIC sequences were identical to those of the human FHIT gene (fragile histidine triad gene) (Ohta et al., Cell 84, 587-597, 1996) . The FHIT gene was recently shown to span the chromosome 3pl4,2 fragile site and the renal carcinoma-associated t(3;8) translocation breakpoint (12) and encodes a dinucleoside S^S' ' '-?¹, P³-triphosphate hydrolase. EXAMPLE 2

Aberrant PSAFP-l transcripts in tumor cells

For detection of fusion transcripts involving PSAFP-l sequences, RT-PCR was used. RT-PCR was carried out as described by Krizman and Berget (Nucleic Acids Res. 21, 5198-5202, 1993) . Briefly, cDNA was synthesized from 5 μg of total RNA. Reverse transcription was performed according to the first strand cDNA synthesis protocol from GIBCO/BRL using PSAFP-l-specific cDNA synthesis primer (96C2322) 5' TGC CTG TCT GAG CCT TTT AGG T 3' and HMGIC-specific cDNA synthesis primer (95C3362) 5' TAC AGC AGT TTT TCA CTA 3' . Specific primers used in the various experiments are given in the legend to Figure 3. The PCR products were subcloned in a TA-cloning kit vector and subsequently used for sequencing. Using the appropriate HMGIC- and PSAFP-l- specific primers, RT-PCR resulted in a product of 481 bp (Figure 3A, lane 3) . The reciprocal PSAFP-1/HMGIC transcript was also expressed as indicated by the presence of a 686 bp RT-PCR product (Figure 3A, lane 4) . These results clearly show that the HMGIC and PSAFP-l genes are reciprocal fusion partners in this pleomorphic adenoma resulting in the structural and functional alteration of the PSAFP-l gene.

The detection of HMGIC/PSAFP-1 and PSAFP-l/HMGIC transcripts in the tumor raised the question whether normal transcripts of these genes were also expressed. Results of RT-PCR analysis using the appropriate primer sets (Figure 3) suggested that indeed this was the case. RT-PCR products of 519 bp and 602 bp were detected which correspond to normal HMGIC (Figure 3A, lane 2) and PSAFP-l (Figure 3A, lane 5) transcripts, respectively. Nucleotide sequence analysis revealed that the coding regions of both RT-PCR products had the wild-type configuration in this particular tumor.

Similar analysis of other tumors, including other tumor types (tumors of the lung, breast, head and neck) revealed expression of aberrant PSAFP-l transcripts, indicating that also genetic events other than chromosome translocations can structurally and functionally alter the PSAFP-l gene. EXAMPLE 3

Diagnostic test for tumor cells

A biopsy of a patient having a pleomorphic adenoma was taken. From the material thus obtained total RNA was extracted using the standard TRIZOL™ LS protocol from GIBCO/BRL as described in the manual of the manufacturer. This total RNA was used to prepare the first strand of cDNA using reverse transcriptase (GIBCO/BRL) and an oligo dT(17) primer containing an attached short additional nucleotide stretch. The sequence of the primer used is AAG GAT CCG TCG ACA TC(T)₁₇. RNase H was subsequently used to remove the RNA from the synthesized DNA/RNA hybrid molecule. PCR was performed using an HMGIC gene-specific primer (5 '-end) and a primer complementary to the attached short additional nucleotide stretch. An aliquot of the thus obtained PCR product was analyzed by gel electroforesis; fusion constructs were detected by comparing them with the background bands of normal cells of the same individual . Nucleotide sequence analysis of the products conclusively established and defined the differences between the PCR products of normal and tumor cells.

In an additional experiment, a second round of PCR was performed with the previously obtained PCR product using combinations of appropriate PSAFP-l- and HMGIC-specific primers to detect HMGIC/PSAFP-1 and PSAFP-1/HMGIC transcripts. The sensitivity and specificity of the test was thus significantly improved.

In a similar PCR experimental setting, biopsies from other tumors were also tested. Aliquots of the PCR products obtained using normal and tumor cells of the same individual were compared by gel electroforesis. Nucleotide sequence analysis of the PCR products conclusively established and defined the differences between the PCR products of normal and tumor cells. EXAMPLE 4

The preparation of antibodies against PSAFP-1-encoded proteins

One type of suitable molecules for use in diagnosis and therapy are antibodies directed against the PSAFP-l gene. Two approaches have been followed to develop them. Based upon the nucleotide sequence of the PSAFP-l cDNA, computer analysis was used to predict the location of antigenic determinants within the proteins. Synthetic peptides containing such determinants were prepared and rabbits were immunized with these. As an alternative approach, cDNA sequences can be expressed in appropriate pro- and eukaryotic expression systems and the polypeptides synthesized in this way can be purified and used for immunization of mice. Cell lines obtained upon transfection or electroporation of constructs of the PSAFP-l gene were helpful in characterizing the antibodies obtained. For the preparation of rabbit polyclonal antibodies directed against the PSAFP-l-encoded proteins, use was made of the following two peptides:

(H-RPVERFHDLRPD) 8-Multiple Antigen Peptide (MAP) and (H-HRNDSIYEELQKHDK) 8-MAP obtained from Research Genetics Inc., Huntsville, AL, USA. The polyclonal antibodies were made according to standard techniques.

Similarly, suitable antibodies were generated using hybrid proteins as antigens. These contained various portions of the PSAFP-l coding region fused in frame to GST (glutathione-S-transferase) . The antibodies that were obtained detected a

17 kDa protein in Western blot analysis of proteins in cells isolated from a wide variety of human tissues, including brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle. Using im unocytochemical analysis, strong nuclear staining and a rather weak cytoplasmic staining was observed. Immunohistochemical evaluation of a variety of human tumors with these antibodies revealed that tumors of some tissue types did not react, including carcinomas of the lung, breast, and colon and squamous cell carcinomas of head and neck.

LEGEND TO FIGURE 3 A: 3' -RACE analysis of total RNA from the pleomorphic adenoma CG592 (lane 1) . PCR products obtained in RT-PCR analysis of total RNA corresponding to a normal HMGIC transcript (lane 2; first round PCR, primers 31970-004 [5-CCC AGC CCT ATC ACC TCA-3'] and 32627-001 [5' -AAG ACC ATG GCA ATA CAG-3'] ; second round PCR, primers 31970-005 [5' -CTC ATC TCC CGA AAG GTG-3'] and 32627-001) , a HMGIC/PSAFP-1 fusion transcript (lane 3; first round PCR, primers 32627-002 [5 ' -ACT GGT TGG CAA TAG CTC TT-3'] and 31970-004; second round PCR, primers 32627-002 and 31970-005) , the reciprocal PSAFP-1/HMGIC fusion transcript (lane 4; first round PCR, primers 96cl578 [5' -ATC CTG GAA GCT TTG AAG CTC A-3'] and 32627-001; second round PCR primers 96C1579 [5* -TCC GTA GT CTA TCT ACA T-3'] and 32627-001), and a normal PSAFP- transcript (lane 5; first round PCR, primers 96C1578 and 96C1581 [5' -TCA CTG GTT GAA GAA TAC AGG-3'] ; second round PCR, primers 96C1579 and 96C1580 [5 '-CAT GCT GAT TCA GTT CCT CTT GG-3'] ) . Lane 6 contains the pGEM DNA markers (M) (Promega) , with sizes of 1,605, 1,198, 676, 517, 460, 396, and 350 bp. B : Nucleotide sequence of the HMGIC/PSAFP-1 fusion transcript detected in tumor CG592. The first three HMGIC exons are fused to exons 9 and 10 of the PSAFP-l gene. The relative positions of the various exons are indicated by arrows above the sequence and those of primers used in nested RT-PCR and 3 ' -RACE reactions are indicated by arrows labelled with the primer number, both above and below the sequence. The stop codon in the HMGIC/PSAFP-1 fusion transcript is indicated by an asterisk.

C: Nucleotide sequence of the reciprocal PSAFP-1/HMGIC fusion transcript. The relative positions of the various exons and primer are indicated as above . As shown, the first 8 exons of the PSAFP-l gene are fused to exons 4 and 5 from HMGIC. The stop codon in the PSAFP-l/HMGIC fusion transcript is indicated by an asterisk

D: Schematic representation of the genomic organization of the HMGIC and PSAFP-l genes showing the hybrid transcripts detected, with fusions occurring in intron 3 of the HMGIC gene and intron 8 of the PSAFP-l gene The relative positions of particular functional domains in the deducted proteins of both genes are linked to the encoding exons. Functional domains: D1-D3, DNA binding domains; S: spacer domain; AD: acidic domain.

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6. Walter, T.A., Xuan Fan, S., Medchill, M.T., Berger, C.S., Decker, H-J.H. and Sandberg, A.A. (1989) . Inv(12) (pll.2ql3) m an endometrial polyp. Cancer Genet. Cytogenet. 41- 99-103.

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8. Mandahl, N. , Orndal, C, Heim, S., Willen, H., Rydholm, A., Bauer, H.C.F. and Mitelman, F. (1993) . Aberrations of chromosome segment 12ql3-15 characterize a subgroup of hemangiopericytomas . Cancer 71: 3009-3013.

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12. Mandahl, N. , Willen, H., Rydholm, A. and Mitelman, F.

(1993) . Rearrangement of band ql3 on both chromosomes 12 in a periosteal chondroma. Genes Chrom. Cancer 6: 121-123.

13. Dal Cin, P., Kools, P., De Jonge, I., Moerman, Ph., Van de Ven W. , Van den Berghe H. (1993a) . Rearrangement of 12ql4-15 in Pulmonary Chondroid Hamartoma . Genes Chrom. Cancer, 8, 131-133.

14. Schoenberg Fejzo, M., Yoon, S.J., Montgomery, K.T., Rein, M.S., Weremowicz, S., Krauter, K.S., Dorman, T.E., Fletcher, J.A., Mao, J., Moir, D.T., Kucherlapati, R.S., and Morton, CC. (1995) . Identification of a YAC spanning the translocation breakpoints in uterine leiomyomata, pulmonary chondroid hamartoma and lipoma. Physical mapping of the 12ql4-15 breakpoint region in uterine leiomyomata. Genomics 26: 265-275.

15. Birdsal, S.H., MacLennan, K.A. and Gusterson, B.A. (1992) . t (6;12) (q23;ql3) and t (10; 16) (q22;pll) in a phyllodes tumor of the breast. Cancer Genet. Cytogenet. 60: 74-77.

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24. Schoenmakers, H.F.P.M., Mols, R. , Wanschura, S., Koolε, P.F.J., Geurts, J.M.W., Bartnitzke, S., Bullerdiek, J., Van den Berghe, H., and Van de Ven, W.J.M. (1994b) . Identification, molecular cloning and characterization of the chromosome 12 breakpoint cluster region of uterine leiomyomas. Genes Chrom. & Cancer. 11: 106-118.

25. Van de Ven, W.J.M. , Schoenmakers, H.F.P.M., Wanschura, S., Kazmierczak, B., Kools, P.F.J., Geurts, J.M.W., Bartnitzke, S., Van den Berghe, H., and Bullerdiek, J.

(1995) . Molecular characterization of MAR, a multiple aberration region on human chromosome segment 12ql3-ql5 implicated in various solid tumors. Genes Chrom. & Cancer. 12: 296-303. (Enclosed as "ANNEX 1")

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41. Geurts, J. , Schoenmakers H.F.P.M., Mols, R. , and Van de Ven, W.J.M. (1994) . Improved procedure for rapid isolation and sequencing of DNA insert termini in yeast artificial chromosomes. Meth. Mol. Cell. Biol., 4: 257- 265.

42. Kools, P.F.J., Wanschura, S., Schoenmakers, E.F.P.M., Geurts, J.W.M., Mols, R., Kazmierczak, B., Bullerdiek, J., Van den Berghe, H. and Van de Ven, W.J.M. (1995) . Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t(l;12) (p22;ql5) as the sole cytogenetic abnormality. Cancer Genet. Cytogenet., 79: 1-7.

(Enclosed as "ANNEX 2")

43. Kazmierczak, B. , Wanschura, S. , Rosigkeit, J. , Meyer- Bolte, K. , Uschinsky, K., Haupt , R. , Schoenmakers, E.F.P.M., Bartnitzke, S., Van de Ven, W.J.M. and Bullerdiek, J. (1995) . Molecular characterization of 12ql4-15 rearrangements in three pulmonary chondroid hamarto as. Cancer Res., in press.

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Claims

1. Multi-tumor Aberrant Growth (MAG) gene designated "PSAFP-l" gene and having essentially the coding part of the cDNA sequence as depicted in figure IA, or the complementary strand thereof, including modified or elongated versions of both strands.

2. Derivatives of the gene as claimed in claim 1, or of its immediate vicinity for use in diagnosis and the preparation of therapeutical compositions, wherein the derivatives are selected from the group consisting of sense and anti-sense cDNA or fragments thereof, transcripts of the gene or practically usable fragments thereof, antisense RNA, fragments of the gene or its complementary strand, proteins encoded by the gene or fragments thereof, antibodies directed to the gene, the cDNA, the transcript, the protein or the fragments thereof, as well as antibody fragments.

3. In situ diagnostic method for diagnosing cells having a non-physiological proliferative capacity, comprising at least some of the following steps: a) designing a set of nucleotide probes based on the information obtainable from the nucleotide sequence of the PSAFP-l gene as claimed in claim 1 or 2, wherein one of the probes is hybridisable to a region of the aberrant gene substantially mapping at the same locus as a corresponding region of the wildtype gene and the other probe is hybridisable to a region of the aberrant gene mapping at a different locus than a corresponding region of the wildtype gene; b) incubating one or more interphase or metaphase chromosomes or cells having a non-physiological proliferative capacity, with the probe under hybridising conditions; and c) visualising the hybridisation between the probe and the gene .

4. Method of diagnosing cells having a non- physiological proliferative capacity, comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) isolating a suitable PSAFP-l gene-related macromolecule therefrom; c) analysing the macromolecule thus obtained by comparison with a wildtype reference molecule preferably from the same individual .

5. Method as claimed in claim 4, comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) extracting total RNA thereof; c) preparing at least one first strand cDNA of the mRNA species in the total RNA extract, which cDNA comprises a suitable tail; d) performing a PCR and/or RT-PCR using a PSAFP-l gene specific primer and a tail-specific and/or partner- specific/nested primer in order to amplify PSAFP-l gene specific cDNA's; e) separating the PCR products on a gel to obtain a pattern of bands; f) evaluating the presence of aberrant bands by comparison to wildtype bands, preferably originating from the same individual .

6. Method as claimed in claim 4, comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) isolating total protein therefrom; c) separating the total protein on a gel to obtain essentially individual bands and optionally transferring the bands to a Western blot; d) hybridising the bands thus obtained with antibodies directed against a part of the protein encoded by the remaining part of the PSAFP-l gene and against a part of the protein encoded by the substitution part of the PSAFP-l gene; e) visualising the antigen-antibody reactions and establishing the presence of aberrant bands by comparison with bands from wildtype proteins, preferably originating from the same individual .

7. Method as claimed in claim 4 , comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) isolating total DNA therefrom; c) digesting the DNA with one or more so-called

"rare cutter" restriction enzymes; d) separating the digest thus prepared on a gel to obtain a separation pattern; e) optionally transfering the separation pattern to a Southern blot; f) hybridising the separation pattern in the gel or on the blot with one or more informative probes under hybridising conditions; g) visualising the hybridisations and establishing the presence of aberrant bands by comparison to wildtype bands, preferably originating from the same individual.

8. Method as claimed in claim 4, comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) isolating total DNA therefrom; c) carry out mutation detection analysis to evaluate gene inactivation using standard technologies as described in "Laboratory Protocols for Mutation Detection" . Ulf Landegren, Oxford University Press, 1996, ISBN 0 19 857795 8.

9. Method as claimed in claim 4, comprising at least some of the following steps: a) taking a biopsy of the cells to be diagnosed; b) extracting mRNA therefrom; c) establishing the presence or the (relative) quantity of mRNA derived from the PSAFP-l gene; and d) comparing the result of step c) with the result of a similar experiment with wildtype cells, preferably originating from the same individual.

10. Method as claimed in any one of the claims 4-

9, wherein the cells having a non-physiological proliferative capacity are selected from the group consisting of the mesenchymal tumors hamartomas (e.g. breast and lung) , adipose tissue tumors (e.g. lipomas), pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of the endometrium, atherosclerotic plaques, and other benign tumors as well as various malignant tumors, including but not limited to sarcomas (e.g. rhabdo yosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid), and haematological malignancies, like leukemias and lymphomas.

11. Anti-sense molecules of a PSAFP-l gene as claimed in claim 1 for use in the treatment of diseases involving cells having a non-physiological proliferative capacity by modulating the expression of the gene.

12. Expression modulators, such as inhibitors or enhancers, including ribozymes, of the PSAFP-l gene as claimed in claim 1 for use in the treatment of diseases involving cells having a non-physiological proliferative capacity.

13. Antisense RNA molecules complementary to the mRNA molecules of the PSAFP-l gene and/or antibodies directed against the gene product of the PSAFP-l gene as claimed in claim 1 for use in the treatment of diseases involving cells having a non-physiological proliferative capacity.

14. Diagnostic kit for performing the method as claimed in claim 3, comprising a suitable set of labeled nucleotide probes.

15. Diagnostic kit for performing the method as claimed in claim 5, comprising a suitable set of labeled probes .

16. Diagnostic kit for performing the method as claimed in claim 6, comprising a suitable set of labeled PSAFP-l gene specific and tail specific PCR primers.

17. Diagnostic kit for performing the method as claimed in claim 7, comprising a suitable set of labeled probes, and suitable rare cutting restriction enzymes.

18. Pharmaceutical composition to replace defective PSAFP-l gene, comprising one or more of the derivatives as claimed in claims 1 and 2.

19. Pharmaceutical composition for lowering the expression level of the PSAFP-l gene in cells having a non- physiological proliferative capacity, comprising one or more of the derivatives as claimed in claim 2 and/or one or more expression modulators as claimed in claim 12.

20. Pharmaceutical composition as claimed in claim 18 or 19, wherein the cells having a non-physiological proliferative capacity are selected from the group consisting of the mesenchymal tumors hamartomas (e.g. breast and lung) , adipose tissue tumors (e.g. lipomas) , pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of the endometrium, atherosclerotic plaques, and other benign tumors as well as various malignant tumors, including but not limited to sarcomas (e.g. rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid) , and haematological malignancies, like leukemias and lymphomas.

21. Use of the derivatives as claimed in claim 2 for the preparation of a diagnostic kit or a pharmaceutical composition for the diagnosis or treatment of diseases or disorders involving cells having a non-physiological proliferative capacity.

22. Use of the expression modulators as claimed in claim 11 for the preparation of a pharmaceutical composition for the treatment of diseases or disorders involving cells having a non-physiological proliferative capacity.

23. Use as claimed in claim 21 or 22, wherein the cells having a non-physiological proliferative capacity are selected from the group consisting of the mesenchymal tumors hamartomas (e.g. breast and lung) , adipose tissue tumors

(e.g. lipomas) , pleomorphic salivary gland adenomas, uterine leiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of the endometrium, atherosclerotic plaques, and other benign tumors as well as various malignant tumors, including but not limited to sarcomas (e.g. rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung, skin, thyroid) , and haematological malignancies, like leukemias and lymphomas.

24. Animal model for the assessment of the utility of compounds or compositions in the treatment of diseases or disorders involving cells having a non-physiological proliferative capacity, which animal is a transgenic animal harbouring a PSAFP-l gene in its genome.

25. Animal model as claimed in claim 24, wherein the PSAFP-l gene is an aberrant PSAFP-l gene, such as a fusion product of the remaining part of the gene and the substitution part of its translocation partner.

26. Animal model as claimed in claim 24, wherein the PSAFP-l gene shows a non-physiological expression level.

27. Animal model for the assessment of the utility of compounds or compositions in the treatment of diseases or disorders involving cells having a non-physiological proliferative capacity, which animal harbours a specific genetic aberration affecting the PSAFP-l gene as claimed in claim 1 in the genome of at least part of its cells, which aberration is induced via homologous recombination in embryonic stem cells.

28. Animal model as claimed in any one of the claims 24-27, which animal is a mammal, in particular a mouse, rat, dog, pig or higher primate, like chimpanzee.

29. Poly- or oligonucleotide probes and primers as disclosed in the description and figures.