Acute Myeloid Leukemia (AML)

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Leukemia (2002) 16, 2078–2083  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Acute myeloid leukemia (AML) having evolved from essential thrombocythemia (ET): distinctive chromosome abnormalities in patients treated with pipobroman or hydroxyurea P Bernasconi1, M Boni1, PM Cavigliano1, S Calatroni1, E Brusamolino1, F Passamonti1, G Volpe2, A Pistorio3, I Giardini1, B Rocca1, M Caresana1, M Lazzarino1 and C Bernasconi1

1Department of Blood, Heart and Lung Medical Sciences of the University of Pavia and Division of Hematology, Policlinico San Matteo IRCCS, Pavia, Italy; 2Department of Clinical and Biological Sciences of the University of Turin, Azienda Ospedaliera San Luigi di Orbassano, Torino, Italy;and 3Scientiﬁc Direction, Clinical Epidemiology and Biometry Unit, Policlinico San Matteo IRCCS, Pavia, Italy

ET is a chronic myeloproliferative disorder rarely evolving into alkylating agents.7 In 1998, however, concern also arose AML, sometimes preceded by a myelodysplastic syndrome about the long-term safety of HU treatment: it was reported (MDS). Such transformations mostly occur in patients treated 32 that 13% of HU-treated ET patients developed MDS/AML or with radiophosphorous ( P) or alkylating agents, especially 8 busulfan. Recently, concern has also arisen about the long- solid tumors. Interestingly, most of these patients carried a term safety of hydroxyurea (HU). Pipobroman (PI), a well toler- 17p deletion (17p−). This chromosome abnormality, due to ated and simple to use drug, constitutes a valid alternative to unbalanced translocations or to monosomy 17 or to i(17q), is those cytoreductive treatments. The present study reports on associated with the presence of pseudo Pelger-Hue´t hypolob- 155 ET patients treated at our institution from 1985 to 1995, and ulation and small vacuoles in neutrophils and with TP53 monitored until December 2000. A good control of throm- − bocytosis was achieved with PI as the only treatment in 106 mutation. It was suggested that 17p identifies a new and spe- patients and with HU in 23 patients. Twenty-six patients cific morphologic–cytogenetic–molecular entity within the received no treatment. After a median follow-up of 104 months, group of secondary MDS/AMLs. In contrast, only anedoctal seven patients (four treated with HU, and three with PI) cytogenetic information is available for ET patients treated developed AML whereas one patient treated with PI developed with pipobroman (PI) alone who developed MDS/AML.9 MDS. A significant difference in progression-free survival was Here we review cases of MDS/AML occurring in 155 ET observed between HU- and PI-treated patients (P = 0.004). A short-arm deletion of chromosome 17 was most frequently patients diagnosed and cytogenetically studied at our insti- detected in HU-treated patients, while a long-arm trisomy of tution from 1985 to 1995 and monitored until December chromosome 1 and a monosomy 7q were seen in PI-treated 2000. Eight cases of MDS/AML were observed: four in patients patients. No TP53 mutation was discovered in the six patients treated with HU and four in patients treated with PI. studied (two HU-treated and four PI-treated). We conclude that these cytogenetic abnormalities are not linked to the natural history of the disease, but rather that they might be induced by the cytoreductive treatment. Materials and methods Leukemia (2002) 16, 2078–2083. doi:10.1038/sj.leu.2402638 Keywords: cytogenetics; FISH; chromosome abnormalities; 17p−; Diagnosis TP53 All 155 patients were diagnosed at the Division of Hematol- ogy, Policlinico San Matteo IRCCS, Pavia, from January 1985 to December 1995. ET was diagnosed according to the Poly- cythemia Vera Study Group (PVSG) criteria.10 A bone marrow Introduction core biopsy was part of the diagnostic work-up in each patient. Essential thrombocythemia is a chronic myeloproliferative disorder with a relatively benign clinical course. However, some patients are asymptomatic while others experience life-threat- ening thromboembolic episodes.1,2 Among these last patients, Treatments cytoreduction is mandatory in those above 60 years of age and those with a platelet count above 1000 × 109/l.3 The drug Cytoreductive therapy was started when the patient met one preferentially given to achieve this goal is hydroxyurea (HU), or all of the following criteria: platelet count greater than 1 × because of its high effectiveness in reducing the incidence of 1012/l, age Ͼ60, previous thrombosis. In most patients induc- thrombotic complications and its low leukemogenic poten- tion treatment consisted of PI, given at the dose of 1 mg/kg/day tial.4 Other myelosuppressive treatments consisting of radi- orally until response. The maintenance dose varied between ophosphorous (32P) or alkylating agents (especially busulfan 0.3 and 0.6 mg/kg/day, according to response. In another (BU)) have also been used, but it has been reported that they smaller group of patients, induction treatment consisted of HU may increase the risk of developing acute myeloid leukemia with a starting dose of 15 mg/kg/day until response. Response (AML).5,6 In fact, until recently, progression of ET to AML, pre- to treatment was defined as a reduction of platelets to 400 × ceded or not by myelodysplasia (MDS), has been observed in 109/l. No patients initially treated with PI or HU received both 3% to 4% of patients, mostly those treated with (32P) or drugs or any other drugs at any time during the course of the disease. No fixed criteria were followed to administer antiaggregat- Correspondence: P Bernasconi, Divisione di Ematologia, Policlinico ing agents, but every patient received antiaggregating therapy San Matteo, IRCCS, 27100-Pavia, Italy; Fax: 39 0382 502250 with aspirin, dypiridamole, or ticlopidine at the onset of the Received 12 April 2001; accepted 5 May 2002 disease. Karyotype abnormalities in MDS/AML having evolved from ET P Bernasconi et al 2079 Follow-up test. In order to control for potential confounding elements, we next constructed a multivariate poisson regression model. Patients were seen at our out-patient clinic every year, unless The following variables were first introduced into the model: any disease complication had occurred. MDS and AML were sex (male or female), age group (less than or greater/equal to diagnosed according to French–American–British (FAB) 65 years of age), type of treatment (HU or PI), treatment dur- criteria. ation (less than or greater/equal to 9 years). A Poisson regression model was fitted and the likelihood-ratio test was used to determine if there was a statistical significant differ- Cytogenetics, fluorescence in situ hybridization (FISH) ence in the likelihood of estimated models. Incidence rate and reverse transcription polymerase chain reaction ratios (IRR) and their 95% confidence intervals (CI) for the best (RT PCR) fit model have been reported. All the statistical tests were two-sided; a P value less then Cytogenetic studies were carried out on bone marrow cells at 0.05 was considered as statistically significant. Statistical univ- diagnosis and during the follow-up, using a trypsin-Giemsa ariate analysis was performed on Statistica (StatSoft Inc., banding technique. Metaphase cells were examined from release 5.5; Tulsa, OK, USA) and multivariate analysis on Stata short-term unstimulated cultures. Chromosome abnormalities (Stata Corp, release 7.0, TX, USA) software packages. were defined according to ISCN.11 FISH, aimed at confirming the short-arm deletion of chromosome 17, was performed on cytogenetic preparations as Results previously reported.12 A digoxygenated p53 DNA probe (17p13.1) (red signal) was applied simultaneously with a bioti- Clinical and hematological features of MDS/AML after nylated alphoid probe specific for the same chromosome ET (green signal) (Oncor, Gaithersburg, MD, USA) and used as an internal control to demonstrate that hybridization had Patients’ clinico–biological features at the onset of the disease effectively taken place. After applying a binomial distri- and treatments are listed in Table 1. After a median follow-up bution,12 the cut-off value for p53 interphase FISH was fixed of 104 months (range 8–240) eight of the 155 patients (5.1%) at 0.8% for p53 monosomy and at 0.3% for p53 trisomy as progressed to MDS/AML. Their clinico–biological features are the highest number of cells lacking a p53 signal was three in summarized in Table 2. They included five women and three normal subjects, probably because of a hybridization failure men with a median age of 56 years (range 40–78). Four and as the highest number of cells with three p53 signals was patients received HU for 53, 94, 47 and 96 months, respect- one in the same subjects. After examining 200 cells from ten ively, while four received PI for 36, 53, 83 and 113 months, normal controls12 the cut-off value for p53 interphase FISH respectively. The median interval between ET diagnosis and was fixed at 4% by adding three times the standard deviation MDS/AML evolution was 89 months (range 47–114). Seven to the mean percentage of cells with two signals due to the patients had AML without a preceeding MDS: one was classi- internal control and only one signal due to the p53 DNA fied as M1, two as M2, three as M4 and one as M7. One probe. The cut-off value for p53 trisomy on interphase cells patient had MDS, subclassified as refractory anemia with was determined in the same way and was fixed at 2%. excess of blasts (RAEB). After the diagnosis of MDS/AML was In order to exclude the presence of a bcr/abl transcript, an made, six patients were given intensive chemotherapy and RT PCR assay was carried out at diagnosis for every patient two monochemotherapy with 6-thioguanine. A short-term diagnosed from 1994 onwards and during follow-up in all partial response with 50% reduction of marrow blasts was others in whom the clinical diagnosis was made before 1994. achieved in only two patients, both treated with intensive The assay was performed as already described.13 chemotherapy.

Polymerase chain reaction (PCR)-single strand Table 1 Clinical–biological features and treatment in the 155 conformation polymorphism (SSCP) and DNA direct patients with essential thrombocythemia studied sequencing Number of patients 155

14 Median age (range) 55 years (15–86) Our PCRSSCP assay was carried out as previously reported Sex (female/male) 87/68 and its sensitivity permits the detection of a TP53 mutation Splenomegaly (number) 8 restricted to 5–10% of the cell population examined, as estab- Previous thrombosis 6 lished by control dilutional analysis using a cell line contain- Platelets × 109/l (range) 900 (530–3000) × 9 ing a previously characterized TP53 mutation.15 Samples were White blood cells (WBC) 10 /l 12.9 (10.8–16.9) (range) investigated for mutations in exons 5 to 8. DNA direct sequen- 15 Cytogenetic abnormalities None cing was performed as previously described. Both strands BCR/ABL positive on RT PCR None were sequenced for each DNA fragment analyzed. Abnormal p53 FISH Nonea Treatments: Pipobroman 106 Statistical analysis Hydroxyurea 23 No treatment 26 Median follow-up, months (range) 104 (8–240) Cumulative incidence of disease progression over 5, 10 and 15 years and incidence rates (person/year) have been calcu- aNormal p53 metaphase and interphase FISH in 11 patients studied lated and reported for each group of patients. Initially, we during disease outcome (2 treated with PI, who subsequently drew the survival curves and compared the differences developed AML, one treated with HU, who subsequently evolved between different groups of patients by means of the log-rank into AML and 8 untreated who never developed AML).

Leukemia Karyotype abnormalities in MDS/AML having evolved from ET P Bernasconi et al 2080 Table 2 Clinical–biological features of the eight ET cases which evolved into MDS/AML

Patient Sex/Age Interval Treatment MDS/AML Karyotype on MDS/AML p53 FISH Pelger- Survival between ET given FAB subtype progression Hue´t (months) diagnosis ( months) anomaly and MDS/AML (months)

1 RP M/57 108 HU: 53 AML-M2 46,XY,del(5)(12q33),del(12)(p11), 58% of cells with only one Yesa 1† del(17)(p11) signal 2 OC F/78 94 HU: 93 AML-M4 46,XX,del(5)(q12q33),del(17)(p11) 64% of cells with only one Yes 9† signal 3 BG F/58 47 HU: 46 AML-M1 46,XX,del(5)(q12q33),+8,del(17) 73% of cells with only one Yes 1† (p11) signal 4 MC F/52 96 HU: 96 AML-M7 46,XX,t(3;6)(q21;p22)/48,XX,idem, 78% of cells with three Noa 3† −5,del(7)(q22),del(9)(q22),add(16) signals (p13),+17,add(17)(p13),+mar,+ring 5 PL F/73 69 PI: 36 AML-M4 48,XX,+1,dic(1;7)(p11;q11),+8,+21 Normal Noa 2† 6 ME F/72 55 PI: 53 AML-M2 46,XX/49,XX,+1,add(1)(p11),−6,−12, Normal Noa 3† +mar1,+mar2 7 GL M/55 84 PI: 83 AML-M4 47,XY,+1,dic(1;7)(p11;q11),+8, Normal Noa 9† del(12)(p12) 8 BE M/40 114 PI: 113 RAEB 46,XY/46,XY,t(1;3)(p36;q21) Normal Noa 14→

F, female; M, male; HU, hydroxyurea; PI, pipobroman. aCases without any TP53 mutation on DNA PCR-SSCP, →, alive; †, dead.

Cytogenetics, FISH and PCR-SSCP result

Chromosome analysis was successful in all eight MDS/AML patients. A mixture of karyotypically normal and abnormal cells was observed in two, a completely abnormal karyotype in six (Table 2). Two patients showed two chromosome defects, while the remaining six had a complex cytogenetic pattern (>three abnormalities). A rearrangement of the short arms of chromosome 17 was detected in all four patients previously treated with HU. Three of them showed a 17p deletion, which was conﬁrmed by FISH with the p53 DNA probe on both metaphase and interphase cells (58–78% abnormal nuclei). The DNA PCR-SSCP assay revealed the absence of any TP53 mutation/overexpression in the only patient studied (no material was available for the other two Figure 1 Proportion of patients surviving without progression by patients). Morphologically, all three patients displayed the Kaplan–Meier. pseudo Pelger-Hue´t hypolobulation and small vacuoles in at the TP53 gene. Morphologically none of them showed the least 5% of neutrophils. Pelger-Hue´t anomaly. The remaining patient presented two cell lines: one carrying a t(3;6)(q26;p22) translocation, the other, without the translocation, having a complex karyotype. In this patient, FISH with Statistical analysis the p53 DNA and the 17␣ probes demonstrated two red and The proportion of patients surviving without progression by two green signals (two normal chromosomes 17) in 68% of Kaplan–Meier is shown in Figure 1. A statistically signiﬁcant the cells examined, and three red and three green signals in difference was observed between HU- vs PI-treated patients the remaining 32%. In these cells the third red and green spots = were localized on add(17)(p13). In all four patients the (P 0.04). Cumulative incidences of disease progression in rearrangement of number 17 was never a unique abnormality; patients treated with HU vs PI are reported in Table 3. The it was often associated with an interstitial long-arm deletion Table 3 Cumulative incidence of disease progression over 5, 10 of chromosome 5. Morphologically the patient did not show and 15 years and incidence rate (person/year) the Pelger-Hue´t anomaly. All four PI-treated patients presented an abnormal karyotype Cumulative Incidence Incidence rate with chromosomes 1 and 7 most frequently involved in unbal- (person/year) anced rearrangements. In particular, two patients showed an Over 5 yrs Over 10 yrs Over 15 yrs unbalanced translocation dic(1;7), one presented a rearrangement of band 1p11 and a trisomy 1q, the last one a translo- HU 8.09% 15.53% 22.37% 0.016884 cation t(1;3). In all these patients, FISH with the p53 DNA PI 1.97% 3.89% 5.78% 0.003972 probe gave normal results on both metaphase and interphase cells and DNA PCR-SSCP assay demonstrated no mutation of Yrs, years; HU, hydroxyurea; PI, pipobroman.

Leukemia Karyotype abnormalities in MDS/AML having evolved from ET P Bernasconi et al 2081 incidence rate (person/year) was 0.016 vs 0.003. IRR and 95% a bromide derivative of piperazine which, although its for- CI for the best fit models are reported in Table 4. The likeli- mula is close to that of alkylating agents, appears to act mainly hood ratio test was statistically significant (P = 0.019) only as a metabolic competitor of pyrimidine bases. It inhibits the when HU- vs PI-treated patients were examined. activity of DNA and RNA polymerases. PI has not been exten- sively employed in ET; on the contrary the notion of its effectiveness in polycythemia vera (PV) goes back to the 1980s.26,27 Discussion In the present study, two PI-treated patients carried the unbalanced translocation t(1;7)(p11;q11), that should always be In our ET patients, cytogenetically and molecularly studied defined as dic(1;7)(p11;q11) on the basis of FISH analysis.28 from the onset of disease and with a median follow-up of 104 Dicentric chromosomes normally result in the loss of the arms months (range 8–240), a clinical evolution into MDS/AML reciprocal to those forming the dicentric region. This does not occurred with an incidence of 5.1%, similar to that reported apply to dic(1;7) which is associated with two apparently nor- in the literature.5–8,16,17 mal chromosomes 1 and results in a trisomy for the long arm To the best of our knowledge, this is the first cytogenetic of chromosome 1(+1q) and in a monosomy for the long arm study dealing with MDS/AML in ET patients previously treated of chromosome 7(7q−) as seen in our two patients. A recent with PI. Until now such an evolution has been described study has demonstrated that dic(1;7) is significantly more mostly in patients who had been given 32P or alkylating common in secondary MDS/AML than in de novo cases, and agents, especially busulfan.6,7 Recently, it has also been is significantly related to previous treatment with alkylating reported that long-term treatment with HU may favor an evol- agents.29 The time range from the exposure to mutagenic ution of ET into MDS/AML,8 marked by a 17p deletion (17p−) agents to the development of MDS/AML is usually between 2 with loss of one TP53 allele, vacuolated neutrophils and pres- and 14 years.30 Our patients received PI for 36 and 83 months entation of the Pelger–Hue´t anomaly. Even if this reported and the time interval from ET diagnosis to MDS/AML develop- association still needs to be proved,18,19 four of our eight ment was 69 and 84 months. Therefore, both might well be AMLs having evolved from ET occurred in subjects treated therapy-related AMLs. However, a recent large cytogenetic with HU and three of them developed a 17p−. In every case study of 165 patients affected by myelofibrosis with myeloid FISH with the p53 DNA probe detected only one red signal, metaplasia has revealed that the dic(1;7) was relatively fre- confirming loss of one TP53 allele. A DNA PCR-SSCP assay quent (7% of all abnormal cases).31 None of the patients in for the TP53 mutation, carried out in one of the three patients the latter series received treatment with PI. It is therefore not (no material was available in the other two cases), detected unexpected to detect such an abnormality in clonally evolved no mutation, despite the fact that the subjects presented hypo- ET. In fact, a dic(1;7) was previously reported in a chemo- lobulated neutrophils and a complex karyotype with 17p and therapy naive patient with ET which evolved first in myelo- 5q deletions. These last biological features are all typically fibrosis and then to acute leukemia (M0).32 However, none of associated with TP53 mutation/overexpression20,21 and sug- our dic(1;7) patients presented a phase of myelofibrosis pre- gest a possible cooperation between TP53 inactivation and an ceding AML development. A third patient carried a trisomy as yet unidentified gene residing within band 5q13.3.22 The 1q, due to +add(1)(p11), and the fourth presented a biological features of our patient are similar to those already t(1;3)(p36;q21). reported in two other previously described patients with no The concern over drug leukemogenicity into ET is unlikely TP53 mutation23 and in a more recent one having an apparent to be resolved in the absence of a prospective study because 17p13 loss but a wild-type TP53 gene.21 leukemic transformation into ET is a time-dependent variable In the fourth patient, cytogenetics and FISH identified three and it is difficult to identify an appropriate control group for a chromosomes 17 with three p53 signals in 32% of the cells retrospectively selected study population. There are numerous screened. On PCR-SSCP analysis no TP53 mutation was dis- published reports of leukemic transformation in ET in the covered. Morphologically, the patient did not show the absence of previous cytoreductive therapy.33 Furthermore, Pelger-Hue´t anomaly. As far as we know such a cytogenetic treatment-unrelated leukemic transformations have previously pattern has been observed in only one other patient having been associated with complex cytogenetic abnormalities.24,34 an AML following untreated ET. Also, this subject did not Therefore, in this clinical setting it is difficult to differentiate show any peculiar morphological or immunophenotypic a natural sequela from a treatment-related event. However, in characteristics.24 the present series none of our untreated patients experienced Few reports in the literature have dealt with MDS/AML in a progression into MDS/AML. ET patients treated with PI alone.10,25 Moreover, in most of The possible association between HU therapy and 17p− them the chromosome pattern has not been investigated. PI is anomaly is still an unanswered question. In fact, an elegant analysis using Sterkers’ own data8 revealed a more impressive association of 17p− with advanced age rather than HU treat- Table 4 Incidence rate ratio (IRR) and 95% confidence interval ment,19 a correlation we were unable to find in our series. (Poisson regression model) Furthermore, the 17p− anomaly was often associated with other complex abnormalities and was not found to be specific IRR 95% CI P (likelihood-ratio test) to either HU treatment or ET.23 In the latter series of 25 17p− cases, 14 were observed in patients with either ET or PV. Four HU vs PI 6.15 1.4–26.99 0.0198 of these 14 cases never received HU therapy and most of the Age у65 yrs vs Ͻ65 yrs 3.66 0.83– 0.1091 10 cases who had prior exposure to HU were also treated 16.05 32 Treatment duration у9 yrs 0.23 0.05–1.23 0.0764 with other agents including PI and P. vs Ͻ9 yrs As far as PI is concerned, a study on ET patients detected an incidence of progression into MDS/AML of 0% and 16%, CI, confidence interval; HU, hydroxyurea; PI, pipobroman; yrs, respectively, when PI was used alone or after the use of other years. agents (usually HU).8 In a prospective report on PV patients,

Leukemia Karyotype abnormalities in MDS/AML having evolved from ET P Bernasconi et al 2082 comparing PI with HU, the leukemogenic risk was approxi- disorders: actuarial probability and main characteristics in a series mately 10% at the 13th year, with no significant difference of 218 patients. Acta Haematol 1991; 85: 124–127. between the two drugs.35 In the present study, the incidence 6 Chistolini A, Mazzucconi MG, Ferrari A, La Verde G, Ferrazza G, Dragoni F, Vitale A, Arcieri R, Mandelli F. Essential thrombocy- of MDS/AML in ET patients treated with PI alone was 3.7%, themia: a retrospective study on the clinical course of 100 patients. while that in ET patients treated with HU alone was mislead- Haematologica 1990; 75: 537–540. ing, since it was unusually high, possibly because of the small 7 Van de Pette JEW, Prochazka AV, Pearson TC, Singh AK, Dickson number of patients. In fact, in studies on a significant number ER, Wetherley-Mein G. Primary thrombocythaemia treated with of patients with a similar follow-up duration or receiving HU busulfan. Br J Haematol 1986; 62: 229–237. ¨ for over 10 years, an evolution into MDS/AML was observed 8 Sterkers Y, Preudhomme C, Lai J-L, Demory J-L, Caulier M-T, Wat- 8,36 tel E, Bordessoule D, Bauters F, Fenaux P. Acute myeloid leukemia in only 3.5% or in none of the patients, respectively. In the and myelodysplastic syndromes following essential thrombocy- present study, we observed a significant difference in themia treated with hydroxyurea: high proportion of cases with progression-free survival between HU- and PI-treated patients 17p deletion. Blood 1998; 91: 616–622. (P = 0.04), with a cumulative incidence higher in HU-treated 9 Messora C, Bensi L, Vanzanelli P, Temperani P, Carotenuto M, patients. Furthermore, the relative risk of developing Sacchi S. Myelodysplastic transformation in a case of essential MDS/AML was six times higher in patients treated with HU thrombocythemia treated with pipobroman. Haematologica 1996; compared to those who received PI. 81: 51–53. − 10 Murphy S, Peterson P, Iland H, Laszlo J. Experience of the polycy- In conclusion, our study suggests that 17p , trisomy 1q and themia vera study group with essential thrombocythaemia: a final monosomy 7q are not related to the natural history of ET and report on diagnostic criteria, survival, and leukemic transition by to its genetic instability, but rather that they might be induced treatment. Semin Hematol 1997; 34: 29–39. by the type of cytoreductive therapy given. A 17p deletion 11 ISCN (1995) Mitelman F (ed). An International System for Human was discovered only in HU-treated patients and it developed Cytogenetic Nomenclature. S Karger: Basel, 1995. solely at the time of disease evolution, similar to what was 12 Bernasconi P, Astori C, Cavigliano PM, Boni M, Malcovati L, Cala- 9 troni S, Caresana M, Bernasconi C. Cytogenetic and FISH analyses reported in another study. A more recent report has further in five patients with hypoplastic bone marrow. Leukemia 2000; stressed that TP53 mutations are drug-related and not inde- 14: 1322–1323. pendent of the type of chemotherapy.21 Deletions or loss of 13 Saglio G, Guerrasio A, Tassinari A, Ponzetto A, Zaccaria A, Tes- 5q, of 17p and a complex karyotype were all closely related to toni P, Celso B, Rege-Cambrin G, Serra A, Pegoraro L, Avanzi GC, TP53 mutations and to previous chemotherapy with alkylating Attadia V, Falda M, Gavosto F. Variability of the molecular defects agents, especially cyclophosphamide. Our three 17p− patients corresponding to the presence of a Philadelphia chromosome in presented similar cytogenetic and morphological features human hematologic malignancies. Blood 1988; 72: 1203–1208. 14 Gaidano G, Pastore C, Santini V, Nomdedeu J, Gamberi B, Cap- even though the one patient tested for the TP53 mutation car- ello D, Vischia F, Resegotti L, Mazza U, Rossi Ferrini P, Lo Coco ried only the wild-type gene. A different conclusion was achi- F, Saglio F. Genetic lesions associated with blastic transformation eved by a recent study which detected TP53 mutations in of polycythemia vera and essential thrombocythemia. Genes untreated patients also.14 However, in the present study none Chromosomes Cancer 1997; 19: 250–255. of our untreated patients with a sufficiently long follow-up 15 Gaidano G, Ballerini P, Gong JZ, Inghirami G, Neri A, Newcomb developed MDS/AML. 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