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(C4) Molecular pathology of MDS
Second International Conference on Myelodysplastic Syndromes
6
(C-6)
(C3) CHROMOSOMAL
HAEMATOLOGICAL
DELETIONS MALIGNANCIES.
IN
W.J.R.HIRST, B.CZEPULKOWSKI, G.J.MUFTI. Department of Haematology, King ’B College Hospital, tions
Over
have
London. a 2 year
period,
chromoaomal
dele-
been observed in 54 patients with haematologica.lmalignancies(myeloid;39,lymphoid;l5) using synchronised culture techniques. In 32 cases these were single abnormalities (myeloid;24,lymphoid;B). Two or more deletions occurred in 6 cases. A total of 61 deletions were found (myeloid;45, lymphoid;l6) involving 15 autosomee (myeloid;l4). In all cases these were interstitial. Of the myeloid malignancies 16 cases wereHDS(RA;3,RARS;3,RABB;B,RABB-t;l, unclaasified;l),l0 cases were AML(Ml;l,M2;4, M4;1,M5;2,M7;l,stem cell 1eukaemia;l) and 12 dieorders(PRV;7, myeloproliferative ET;3, unclaseified;2). 69% deletions in the myeloid malignancies occurred in 6 chromosomes (#20;9,#5;5,#17;5,#9;4,#12;4) with consistency of breakpoints in 61% of this group (40% in myeloid group overall). The breakpoints corresponded to or were adjacent to fragile sites in 49% (proximal;64%, distal;33%). These data chromoeomal deletions in emphaeiee that haematological malignancies are interstitial. The high frequency of consistent breakpoints in the more commonly involved chromosomes euggeets that with some deletions the exact breakpoints may be important possibly through juxtaposition of genes rather than loss of critical regions. The data also suggest that there may be different mechanisms for the devthe proximal and the distal elopment of breakpoints.
(C4) MOLECULAR PATHOLOGY OF MDS R.A.Padua, S.A.Ridqe, G.Carter, P.G.Cachia, J-Thomas, E.Thompson, J.Whittaker, A.Jacobs. Department of Haematoloqy, UWCM, Cardiff. Oncoqene activation and monoclonality are characteristics of malignant cells. FMS and RAS oncoqene mutations have been shz to occur in MD.5 and AML with the highest incidence in those cases with a monocytoid Patients in remission from a phenotype. malignancy treated with conventional therapy are at risk of developing secondary MDS and DNA from peripheral blood of such AML. patients have been shown to harbour similar oncoqene mutations and monoclonal haemopoiesis using an X-linked probe M27B. Time from treatment was significant with most mutations detected in patients more than 1000 days post treatment. Whereas an increase in incidence ofmonoclonality intreatedpatients was observed compared to normal females, this did not correlate with time from treatment. These findings indicate the presence of a clonally expanded population of abnormal cells. Continued follow-up of these patients at risk of developing secondary malignancies should enable us to determine if we can predict those who will develop disease.
MUTATIONS OF THE P53 GENE IN MYELODYSPLASTIC SYNDROMES (MDS). A REPORT ON 152 CASES. P. Fanaux, Ph. Jonveaux, I. Quiquandon,J.L.Lai,C. Preudhomma. MU. Loucheux-Lefebvre,F. Sauters. R. Berger, J.P. Kerckaert. INSERM U 124 (Lille).Ll301 (Paris), Francs. Mutationsof the P53 gene, which has been mapped to 17~13, have been increasinglyrrpxted In solidtumors,especiallyin patientswho had lost one P53 allele. Mo8t mutation8ccourred in 4 ‘hotspow. situated between exons 5 and 8. We looked for such mutations In bone marrow samples obtained from 152 oases of MDS at dlagnorlr, lncludlng25 RA 5 RARS, 50 RAE& 15 RAEBT and 57 CM@_ (16therapy related MDS and 136 de now MDS). Successfulcytogenetioanalyrir wu made in 119 cases. Detectionof xorw 5 to 8 was made in the 152 cases by ringle strand the mutatlonr in ?? conformationpolymorphlrm (SSCP) malyois. a sensitivamethod which can ds~~naJnglenucl~tiderubnitutioninaDNAfragmantof 4CObporless amolifled bv PCR. Our orellmlnarvwork on normal DNA and cell linee with kndwnP53 iene mutatidnr of SSCb had shownthe rnritivityand epecificity of SSCP In detecting point mutationsin exons 5 to 8 of the P53 gene. 147 of the 152 ~1801 of MDS had normal SSCP migratingpatterns for axons 5 to 8. An abnormal SSCP pattern was found In 5 patients : for exon 5 in 2 patients, for exon 7 in 2 patlentr, and for axon 8 in one patient. Sequence analysis confirmrdthr preeenoeofapointmutatlonintheoorrespondingexoninevery case, con&sting of a base wbstitution (leading to a change of the encoded aminoacid) In 4 cases, and inaertlonof 2 nucleotlderin the remainingpatient. 3 of the 8 patients with P53 gene mutationhad 17p monosomy, along with several other chromowme rearrangementa. The remaining 2 patienta with P53 mutations alao had complex oytogenetk rearrangements, but without chromosome 17 abnormalities.Only 3 casea of 17p monoaornywerepresent in the 114 karyotyped patientawho had no P53 9ene mutationsin exon 5 to 8.AJlthe8patientewithPS3genemutationrhadadvanoedMDS(RAEB,lVEE T or CMML). Three progressedto AML and survivalwas ehort, ranging from 1 to 15 month8 (median 8). Our findingswggeat that P53 gene mutationsare nafysir 8howr deletion rare in MDS except In patient8 In whom oytogenetic?? of one P53 allele. Becauw all the MDS with PW mutations had advanced disease, with multiple ohromoeomalabnormallUer, the early or late occurdominance of P53 mutation0in patienta with 17p monoeomyfita better with the ?eoeaeive’model of tumor wpprerrlve activityof the P53 gene than with the ‘dominant’model in which alterationof only one allele la auffioientfor the development of malignancy.
(C7) ALTERATIONS IN THE ~53 GENE IN PRIMARY MYELODYSPLASTIC SYNDROME (pMD8) w; H~$~!$l",","~f;~ ;:;;;:&s of Haematology, Ki g's CA11 ge Hospital London, Ankara', Barcelona? , Viennaq , Innsbruck‘4. p53 is a nuclear phosphoprotein expressed at low levels in non-transformed cells and plays a critical role in the transition of cells through the cell cycle. Located on 17~13, alteration of this tumour suppressor gene is emerging as the commonest genetic change in human cancer. Allelic loss, point mutation and gross structural rerrangements are seen in several malignancies and human cell lines including blast crisis CHL and HL60. We performed Southern blot analysis on 52 cases of BUDS; M=37 : F=15; Age 25-S6,median 67 years; RA=lS, RARS=2, RAEB=ll, RAEB-t=4, aCML= 2. Successful cytogenetice was performed in 85% of cases. No 17~ abnormalities were detected. DNA extracted from peripheral blood/marrow was digested with the restriction enzymes HindIII, EcoRI, BamHI and hybridised with a 1.76kb SalIEcoRI cDNA probe pR4.2 (courtesy of Ed Harlow). An abnormal pattern was found in one case (CMML) with Hind111 alone. It is possible that the additional band could represent an RFLP; however a similar pattern was not seen in any of the other cases/normals and it may represent a point mutation. our results suggest that gross structural alterations are uncommon in pMDS. As in other haematological and solid tumours point mutations in the conserved regions of exons 5-S need to be investigated.
6
(C-6)
(C3) CHROMOSOMAL
HAEMATOLOGICAL
DELETIONS MALIGNANCIES.
IN
W.J.R.HIRST, B.CZEPULKOWSKI, G.J.MUFTI. Department of Haematology, King ’B College Hospital, tions
Over
have
London. a 2 year
period,
chromoaomal
dele-
been observed in 54 patients with haematologica.lmalignancies(myeloid;39,lymphoid;l5) using synchronised culture techniques. In 32 cases these were single abnormalities (myeloid;24,lymphoid;B). Two or more deletions occurred in 6 cases. A total of 61 deletions were found (myeloid;45, lymphoid;l6) involving 15 autosomee (myeloid;l4). In all cases these were interstitial. Of the myeloid malignancies 16 cases wereHDS(RA;3,RARS;3,RABB;B,RABB-t;l, unclaasified;l),l0 cases were AML(Ml;l,M2;4, M4;1,M5;2,M7;l,stem cell 1eukaemia;l) and 12 dieorders(PRV;7, myeloproliferative ET;3, unclaseified;2). 69% deletions in the myeloid malignancies occurred in 6 chromosomes (#20;9,#5;5,#17;5,#9;4,#12;4) with consistency of breakpoints in 61% of this group (40% in myeloid group overall). The breakpoints corresponded to or were adjacent to fragile sites in 49% (proximal;64%, distal;33%). These data chromoeomal deletions in emphaeiee that haematological malignancies are interstitial. The high frequency of consistent breakpoints in the more commonly involved chromosomes euggeets that with some deletions the exact breakpoints may be important possibly through juxtaposition of genes rather than loss of critical regions. The data also suggest that there may be different mechanisms for the devthe proximal and the distal elopment of breakpoints.
(C4) MOLECULAR PATHOLOGY OF MDS R.A.Padua, S.A.Ridqe, G.Carter, P.G.Cachia, J-Thomas, E.Thompson, J.Whittaker, A.Jacobs. Department of Haematoloqy, UWCM, Cardiff. Oncoqene activation and monoclonality are characteristics of malignant cells. FMS and RAS oncoqene mutations have been shz to occur in MD.5 and AML with the highest incidence in those cases with a monocytoid Patients in remission from a phenotype. malignancy treated with conventional therapy are at risk of developing secondary MDS and DNA from peripheral blood of such AML. patients have been shown to harbour similar oncoqene mutations and monoclonal haemopoiesis using an X-linked probe M27B. Time from treatment was significant with most mutations detected in patients more than 1000 days post treatment. Whereas an increase in incidence ofmonoclonality intreatedpatients was observed compared to normal females, this did not correlate with time from treatment. These findings indicate the presence of a clonally expanded population of abnormal cells. Continued follow-up of these patients at risk of developing secondary malignancies should enable us to determine if we can predict those who will develop disease.
MUTATIONS OF THE P53 GENE IN MYELODYSPLASTIC SYNDROMES (MDS). A REPORT ON 152 CASES. P. Fanaux, Ph. Jonveaux, I. Quiquandon,J.L.Lai,C. Preudhomma. MU. Loucheux-Lefebvre,F. Sauters. R. Berger, J.P. Kerckaert. INSERM U 124 (Lille).Ll301 (Paris), Francs. Mutationsof the P53 gene, which has been mapped to 17~13, have been increasinglyrrpxted In solidtumors,especiallyin patientswho had lost one P53 allele. Mo8t mutation8ccourred in 4 ‘hotspow. situated between exons 5 and 8. We looked for such mutations In bone marrow samples obtained from 152 oases of MDS at dlagnorlr, lncludlng25 RA 5 RARS, 50 RAE& 15 RAEBT and 57 CM@_ (16therapy related MDS and 136 de now MDS). Successfulcytogenetioanalyrir wu made in 119 cases. Detectionof xorw 5 to 8 was made in the 152 cases by ringle strand the mutatlonr in ?? conformationpolymorphlrm (SSCP) malyois. a sensitivamethod which can ds~~naJnglenucl~tiderubnitutioninaDNAfragmantof 4CObporless amolifled bv PCR. Our orellmlnarvwork on normal DNA and cell linee with kndwnP53 iene mutatidnr of SSCb had shownthe rnritivityand epecificity of SSCP In detecting point mutationsin exons 5 to 8 of the P53 gene. 147 of the 152 ~1801 of MDS had normal SSCP migratingpatterns for axons 5 to 8. An abnormal SSCP pattern was found In 5 patients : for exon 5 in 2 patients, for exon 7 in 2 patlentr, and for axon 8 in one patient. Sequence analysis confirmrdthr preeenoeofapointmutatlonintheoorrespondingexoninevery case, con&sting of a base wbstitution (leading to a change of the encoded aminoacid) In 4 cases, and inaertlonof 2 nucleotlderin the remainingpatient. 3 of the 8 patients with P53 gene mutationhad 17p monosomy, along with several other chromowme rearrangementa. The remaining 2 patienta with P53 mutations alao had complex oytogenetk rearrangements, but without chromosome 17 abnormalities.Only 3 casea of 17p monoaornywerepresent in the 114 karyotyped patientawho had no P53 9ene mutationsin exon 5 to 8.AJlthe8patientewithPS3genemutationrhadadvanoedMDS(RAEB,lVEE T or CMML). Three progressedto AML and survivalwas ehort, ranging from 1 to 15 month8 (median 8). Our findingswggeat that P53 gene mutationsare nafysir 8howr deletion rare in MDS except In patient8 In whom oytogenetic?? of one P53 allele. Becauw all the MDS with PW mutations had advanced disease, with multiple ohromoeomalabnormallUer, the early or late occurdominance of P53 mutation0in patienta with 17p monoeomyfita better with the ?eoeaeive’model of tumor wpprerrlve activityof the P53 gene than with the ‘dominant’model in which alterationof only one allele la auffioientfor the development of malignancy.
(C7) ALTERATIONS IN THE ~53 GENE IN PRIMARY MYELODYSPLASTIC SYNDROME (pMD8) w; H~$~!$l",","~f;~ ;:;;;:&s of Haematology, Ki g's CA11 ge Hospital London, Ankara', Barcelona? , Viennaq , Innsbruck‘4. p53 is a nuclear phosphoprotein expressed at low levels in non-transformed cells and plays a critical role in the transition of cells through the cell cycle. Located on 17~13, alteration of this tumour suppressor gene is emerging as the commonest genetic change in human cancer. Allelic loss, point mutation and gross structural rerrangements are seen in several malignancies and human cell lines including blast crisis CHL and HL60. We performed Southern blot analysis on 52 cases of BUDS; M=37 : F=15; Age 25-S6,median 67 years; RA=lS, RARS=2, RAEB=ll, RAEB-t=4, aCML= 2. Successful cytogenetice was performed in 85% of cases. No 17~ abnormalities were detected. DNA extracted from peripheral blood/marrow was digested with the restriction enzymes HindIII, EcoRI, BamHI and hybridised with a 1.76kb SalIEcoRI cDNA probe pR4.2 (courtesy of Ed Harlow). An abnormal pattern was found in one case (CMML) with Hind111 alone. It is possible that the additional band could represent an RFLP; however a similar pattern was not seen in any of the other cases/normals and it may represent a point mutation. our results suggest that gross structural alterations are uncommon in pMDS. As in other haematological and solid tumours point mutations in the conserved regions of exons 5-S need to be investigated.