in patients with myelodysplasia using archived blood

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1992 79: 1266-1270

Longitudinal analysis of point mutations of the N-ras proto-oncogene in patients with myelodysplasia using archived blood smears H van Kamp, C de Pijper, M Verlaan-de Vries, JL Bos, CH Leeksma, H Kerkhofs, R Willemze, WE Fibbe and JE Landegent

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Longitudinal Analysis of Point Mutations of the N-ras Proto-oncogene in Patients With Myelodysplasia Using Archived Blood Smears By Harmen van Kamp, Christine de Pijper, Mattie Verlaan-de Vries, Johannes L. Bos, Chris H.W. Leeksma, Hans Kerkhofs, Roe1Willemze, Willem E. Fibbe, and James E. Landegent We performed a longitudinal analysis of point mutations of the N-ras proto-oncogene in patients with myelodysplasia and a follow-up of at least 2.5 years after diagnosis. Point mutations at codons 12, 13, and 61 of the N-ras oncogene were analyzed after in vitro amplification of N-ras specific sequences followed by dot-blot hybridization. Lysed cells scraped from archived blood and bone marrow smears were used as template for a polymerase chain reaction. In 3 of 90 patients tested (3.3%), a mutation in codon 12 could be detected in the most recent blood smears. All available blood and bone marrow samples of these patients were subsequently analyzed for the occurrence of that particular muta-

tion. In all three cases the mutation was not detectable at diagnosis, but was acquired later during the course of the disease. In two of these patients this event was associated with rapid deterioration and transformation to acute leukemia. However, the third patient showed a protracted course during a period of 5 years after acquisition of the mutation. These results indicate that activation of the N-ras protooncogene in these three patients represents a secondary phenomenon associated with disease progression in some cases, but compatible with stable disease in others. o 1992 by The American Society of Hematology.

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sent another important event in the development of MDS. In particular, activated ras oncogenes have been found in myelogenous disorders. The family of human ras proto-oncogenes consists of three closely related genes: H-rus, K-ras, and N-rus. They encode homologous GTPase proteins of 21 Kd (p21“) located at the inner surface of the cell membrane. The ras proto-oncogenes acquire their oncogenic potential when point mutations occur at the codons 12, 13, or 61, resulting in a single amino acid substitution in the protein.’ Activation of one of the ras genes has been detected in a variety of human malignancies.’ High incidences are found in adenocarcinoma of the pancreas (90%) and colon (50%). In about 30% of cases with acute myeloid leukemia (AML) point mutations have been found, especially of the N-ras oncogene? An activated ras oncogene has also been reported in 6% to 40% of patients with MDS.”I3 Several studies have shown a correlation between the presence of an N-rus mutation and the risk of developing acute leukemia. From these studies the presence of a mutation has emerged as an indicator of poor prognosis. However, no longitudinal studies have yet been reported to support this notion. In the present study we have used archived blood and bone marrow smears from patients with MDS and a follow-up of at least 2.5 years as a source of DNA for in vitro amplification. Point mutations of the N-rus oncogene were detected by amplifying rus-specific sequences using the polymerase chain reaction (PCR) followed by hybridization with mutation-specific oligomers. In three patients an N-ras mutation was detected in the most recent blood smears. It was noted that these mutations were acquired during the course of MDS. The mutational event was associated with evolution to overt leukemia in two patients. However, the third patient showed a protracted course over several years after activation of the N-ras oncogene.

YELODYSPLASTIC SYNDROMES (MDS) comprise a clinically defined group of disorders characterized by a variable degree of anemia, granulocytopenia, and/or thrombocytopenia. The bone marrow is normocellular or hypercellular and shows maturation defects in one or several lineages of differentiation.’ The French-AmericanBritish (FAB) Cooperative Study Group proposed a classification of MDS into five subgroups based on the number of blast cells in peripheral blood and bone marrow, the presence of ringed sideroblasts in the bone marrow, and the number of peripheral blood monocytes? The FAB subtypes correlate with survival and with the risk of transformation to acute leukemia that occurs in approximately 30% of patients. It has been well known that MDS represent a group of clonal disorders. Cytogenetic abnormalities have been found in 40% to 60% of patients with de novo MDS. Clonal hematopoiesis has also been shown by glucose-6-phosphate dehydrogenase (Gd-PD) isoenzyme analysis3 and studies of patterns of X-chromosome inactivation using restriction fragment length polymorphisms of X-linked genes in conjunction with their methylation ~ a t t e r n .Studies ~ . ~ combining karyotyping and G-6-PD analysis in patients with MDS have led to the hypothesis of a multi-step pathogenesis of this disorder.6 Activation of a proto-oncogene may repreFrom the Laboratory of Experimental Hematology, Department of Hematology, University Medical Center; the Laboratory of Molecular Carcinogenesis, Sylvius Laboratories, University of Leiden, Leiden; and the Department of Hematology, Leyenburg Hospital, The Hague, The Netherlands. Submitted March 18,1991; accepted October 16,1991. Supported by grants from the Dutch Cancer Society (ZKW90-07) and from the J.A. Cohen Institute for Radiopathology and Radiation Protection. Address reprint requests to Harmen van Kamp, MD, Department of Hematology, Bldg I , C2-R, University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 I992 by The American Society of Hematology. 0006-4971I921 7905-0010$3.00/0 1266

PATIENTS AND METHODS

Patients. Stained archived blood smears from 90 patients with a confirmed diagnosis of MDS and with a follow-up of at least 2.5 years (median 4 years, range 2.5 to 20) were analyzed for the presence of point mutations at codons 12, 13, and 61 of the N-ras oncogene. These patients had an initial diagnosis of refractory Blood, Vol79, No 5 (March 1). 1992: pp 1266-1270

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anemia (RA, 24), RAwith ringed sideroblasts (RARS, 33), chronic myelomonocytic leukemia (CMML, 17), RA with excess blasts (RAEB, 4) according to the FAB criteria, and unclassifiable MDS (12) made between 1958 and 1984.14None of the patients were alive at the time of the study. Clinical data were obtained from the patient records (Table 1). Disease progression was defined either by progressive bone marrow failure resulting in an increasing need for red blood cell transfusions (including the need for transfusions in patients who were previously transfusion independent or a doubling of the number of transfusions over a 6-month period), by the occurrence of infections (as documented by positive cultures or fever responding to antimicrobial therapy) and hemorrhage (for which platelet transfusions were indicated), or by an increasing number of immature myeloid cells (blasts) or monocytes in blood and bone marrow resulting in a different FAB subtype. Involvement of the peripheral blood with more than 25% and of the bone marrow with more than 30% myeloblasts was diagnostic for transformation to acute leukemia. Archived blood smears were available with intervals ranging from 1 week to 6 months. For screening purposes, the most recent smears were tested for the presence of an N-ras oncogene mutation. In those cases with a mutation in the screening sample, all blood and bone marrow smears taken during the interval between diagnosis and death were studied. Sample preparation. To obtain DNA for in vitro amplification, cells were scraped from the object glasses and suspended in 200 pL of the PCR buffer that consisted of 10 mmol/L Tris-HC1, pH 8.3, 3.0 mmol/L MgCl,, 50 mmol/L KCI, 0.01% gelatine (wt/vol), to which 0.45% NP40 (vol/vol), 0.45% Tween 20 (vol/vol), and pronase (1 pg/pL; Boehringer, Mannheim, Germany) were added. The samples were incubated overnight at 37"C, heated at 95°C for 10 minutes, and centrifuged at 14,000 rpm. N-ras mutation detection. PCR and subsequent hybridization with mutation-specific oligomers was performed as described b e f ~ r e with ' ~ minor modifications. Briefly, 10 pL of the sample supernatants was added to 80 pL of the PCR buffer containing 25 pmol of each of the amplimers localized upstream and downstream of either the codons 12 and 13, or codon 61 of the N-ras oncogene. After incubation at 95°C for 5 minutes, PCR was initiated by the addition of 2.5 U Taq polymerase (Cetus, Emeryville, CA) in 10 pL buffer, also containing each deoxynucleotide triphosphate (to a final concentration of 0.25 mmol/L each). The amplification consisted of 33 cycles of denaturation (1 minute 95"C), annealing (1.5 minutes, 55"C), and chain elongation (1.5 minutes, 72°C) using a thermocycler (Bio-med, Theres, Germany). The generated DNA fragments of 109 bp (N12/13) or 103 bp (N61) were analyzed by gel electrophoresis. Aliquots of 2.5 pL of the in vitro amplified DNA were spotted onto Nylon filters (Gene Screen Plus; Du Pont, Boston, MA) and cross-linked by illumination with a 254-nm W lamp (1.6 kJ/m2).Filters were hybridized for 1.5 hours in 3.0 mol/L tetramethyl-ammoniumchloride,50 mmol/L Tris-HCI, pH 7.5, 5 mmol/L EDTA, 1% sodium dodecyl sulfate (SDS), and 1% nonfat dry milk (Protifar; Nutricia, Zoetermeer, The Netherlands) at 56°C in the presence of 100 pg of a y'*P-labeled oligonucleotide probe Table 1. Clinical Characteristics Diagnosis

RA RARS RAEB CMML Unclassifiable Total

No. of Patients

Progression

24

18 (75) 16 (48) 2 (50) 7 (41) 6 (50) 49 (54)

33

4 17 12 90

("4

Transformation (04

6 (25) 2 (6) 2 (50) 3 (18)

3 (25) 17 (19)

(specific activity of about lo9 dpm/pg). The probes, all 20-mers, represent the normal codons as well as all possible mutations (17 in total) that have been shown to activate the N-mas gene. The filters were washed for 15 minutes at 60°C in the same buffer containing 0.1% SDS without the addition of nonfat dry milk. In case negative control samples exhibited a considerable background signal, an additional wash of all filters, either hybridized with the wild-typeor the mutation-specific oligonucleotides, was performed in 5X SSPE (1X SSPE = 10 mmol/L sodium phosphate pH 7.0, 0.18 mol/L NaC1, and 1 mmol/L EDTA), 0.1% SDS for 10 minutes at 62°C (N12/13) or 59°C (N61). Filters were exposed to Fuji X-ray films (Tokyo, Japan) at -80°C for 16 hours using intensifying screens. RESULTS

As shown by gel electrophoresis and confirmed by posi-

tive wild-type hybridization signals, the specific fragments containing the codons 12 and 13 and codon 61 of the N-rus oncogene were successfully amplified from the latest available blood smears of all 90 patients with MDS. These samples were screened for ail possible mutations at these codons (17 in total). No mutations in the codons 13 and 61 were detected in any of the patients. However, in three of them (3.3%) a point mutation in codon 12 was found, one resulting in a Gly + Cys and two in a Gly + Asp substitution. The earlier blood and bone marrow smears were subsequently examined for the presence of that particular mutation. The sensitivity of the procedure was determined by detecting the N61-rus (Gln + Leu) mutation in cells from the HL-60 leukemia cell line," titrated against peripheral blood cells from a normal individual. The final concentration of cells was 5 x 106/mL. Five microliters of these samples (ie, 25,000 cells) were smeared onto glass slides. The N-rus mutation could be demonstrated in the samples containing 1% to 5% HL-60 cells, indicating that 250 to 1,250 mutated cells can be detected (data not shown). Patient T.Y. (Fig 1) was a 69-year-old man, when, at the end of 1970, RA was diagnosed. Karyotyping showed a clone with an extra chromosome of the C-group (47,XY,+C; no banding techniques were available at that time). In early 1972 the bone marrow smear showed progression toward RAEB, with 6% myeloblasts. In January 1973 he developed a rapidly progressive leukocytosis (up to 50.0 x 109/L)with the appearance of myeloblasts in the peripheral blood (up to 14.0 x 109/L). Until that time no N-rus mutations were detected in smears from peripheral blood and bone marrow. However, in January 1973 an N12-rus (Gly -+ Cys) mutation was detected. The mutation signal was of a lower intensity than the wild-type signal, indicating that the activated ius gene was present in a subpopulation of cells. The patient died due to recurrent urogenital infections in April 1974. Patient L.B. (Fig 2) was a 63-year-old woman, when in April 1966, RA was diagnosed. Karyotyping was not performed. She showed a protracted course until in 1974 a progression toward CMML occurred, with 7% blast cells in the bone marrow smear. From that moment onward, a steady increase in leukocyte count with progressive monocytosis developed over a period of several years until the bone

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. VAN KAMP ET AL

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-

follow-up (calendar years) marrow smear in 1976 ultimately showed a diagnosis of AML (40% bone marrow blasts). Cytogenetic analysis of bone marrow cells at that time showed an 47,XX, t(6;?)(q13;?) in all metaphases analyzed. At that moment, an N-rus (Gly + Asp) mutation was detected for the first time in peripheral blood and bone marrow cells. The intensity of the mutation signal finally equaled the intensity of the wild-type signal, indicating that in the end on average each cell harbored the mutated N-rus allele in addition to the normal allele. She died in December 1976 due to cerebral infarction.

Patient J.W. (Fig 3) was an 81-year-old woman when, in 1980, the diagnosis of CMML was established. There were no cytogenetic abnormalities. At diagnosis, no point mutation of the N-rus oncogene was found. From 1982 onward, an N12-rus (Gly + Asp) mutation was detected in peripheral blood leukocytes. The mutation signal showed an increasing intensity that coincided with slowly increasing leukocyte and monocyte counts. At the end of 1983 the diagnosis CMML was confirmed by bone marrow cytology. At that time, the N12-rus mutation was also present in the bone marrow cells containing 5% bone marrow blasts. All

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Fig 2. Patient L.B. Number of leukocytes (4-) and monocytes (-04 in peripheral blood cells. PCR-baseddot-blot oligonucleotide hybridization: A, Nl2-res wild-type hybridization signal in samples from peripheral blood (pbl) and bone marrow (bm); E, mutation signal (gly -+ asp) in pbl and bm.

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From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only. 1269

RAS MUTATIONS IN MYELODYSPLASIA

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10 Fig 3. Patient J.W. Number of leukocytes (-O-) and monocytes (-O-) in peripheral blood cells. PCR-baseddot-blot oligonucleotide hybridization:A, NlZ+as wild-type hybridization signal in samples from peripheral blood (pbi) and bone marrow (bm); B, mutation signal (gly asp) in pbl and bm.

-

'80

bone marrow metaphases showed a normal karyotype. At the end of 1987, 5 years after the acquisition of the N-rus mutation, a progression of the disease was noted with rapidly increasing leukocyte counts and progressive monocytosis. The patient died in December 1987 at the age of 89 years. DISCUSSION

This study shows that the small number of cells present on archived smears of peripheral blood and bone marrow contain sufficient amounts of DNA for in vitro amplification and subsequent dot-blot hybridization to analyze the prcsence of point mutations of the N-rus oncogene. In this way, we were able to study patients with MDS for a prolonged period of time. Mutations in the codons 13 and 61 were not detected in any of the samples. In 3 of 90 patients tested (3.3%), an activated N-rus oncogene involving a mutation in codon 12 was found in the most recent blood smears. In these patients the mutation was not detected in the blood and bone marrow smears taken at the time that MDS was diagnosed. In patient T.Y. the clonal nature of the disease was confirmed by the presence of an abnormal karyotype at diagnosis. In the three patients studied, activation of the N-rus oncogene was detectable relatively late during the course of the disease. This does not appear to be a general phenomenon because in a prospective study observing MDS patients up to 18 months Melani et a!" observed an N-rus mutation at diagnosis in 3 of 15 patients. The fact that two of these patients had advanced disease (RAEB and RAEB in transformation) at diagnosis may reflect rapid disease progression in patients with an early or primary activation of the N-rus proto-oncogene. The incidence of N-rus mutations found in the present study was relatively low when compared with other reports" and can be explained by patient selection. We selected

follow-up (calendar years) MDS patients with a long follow-up, which might favor patients with a relatively good prognosis. Patients with CMML appear to have a higher chance of acquiring rus mutations than other patients with MDS.'"I7 In patients with AML rus mutations are also preferentially found in those cases with monocytic involvement (M4 or M5).'"'.'' In accordance, two of our patients with mutations of the rus oncogene were diagnosed as CMML. However, it is not clear whether an N-rus mutation preferentially influences hematopoiesis to myelomonocytic differentiation or whether myclomonocytic cells are more susceptible for acquiring an N-rus mutation. In patients L.B. and J.W. an activated N-rus oncogene was found after a diagnosis of CMML had been established. These data suggest that N-rus mutations are more likely to develop in cells of myelomonocytic differentiation. Activation of the N-rus proto-oncogene was associated with evolution to overt leukemia and a poor prognosis in two (T.Y. and L.B.) of the three patients with an N12-rus mutation. In both patients the first detection of the mutation coincided with rapid progression of the disease after a stable period of 2.5 and 10 years, respectively. In contrast, only 15 of 87 (17%) MDS patients without an N-rus mutation in this study showed transformation to acute leukemia. In accordance, other reports have also shown a relation between the occurrence of activation of the rus oncogene and leukemic transf~rmation.'"~~''.~~ Using the NIH/3T3 transfection assay, Hirai et ala found activated N-rus genes in the bone marrow of three of eight patients with MDS. Two of these patients showed rapid progression to overt acute leukemia. The fraction of cells containing the mutated N-rus oncogenes gradually increased in these patients, concurrently with an increase in the fraction of blast cells.21However, in the present study not only blast cells, but also more mature cells, appeared to contain the

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activated N-ras oncogene because in patients L.B. and J.W. the mutations could also be detected in the blood smears, in which no blast cells were present. In the third patient (J.W.), acquisition of a point mutation of the N-ras protooncogene was not associated with disease progression and was compatible with stable disease for many years. Yunis et all7 found an N-ras mutation at codon 12 or 13 or a K-ras mutation at codon 12 in 11 of 27 patients with primary MDS. They identified two groups of patients with MDS and a mutated ras gene. The first group has incurred an ras mutation at an early stage in a multipotent stem cell and the mutation is present in all blood and bone marrow cells, including mature T lymphocytes? In these patients, the mutation-specific hybridization signal is of equal or nearly equal intensity as the wild-type hybridization signal. The second group of patients has obtained an ras mutation later during the course of the disease, and the cells harboring the mutation represent a newly evolved cell clone. Initially, the mutation-specific signal is weaker than the wild-type signal in these patients. In accordance, in patient T.Y. the mutation represented a late event with the mutation-specific

VAN KAMP ET AL

signal showing less intensity than the wild-type signal, indicating that not all cells were affected by the mutation. In the other patients (L.B. and J.W.) that we studied, the occurrence of N-ras activation also appeared to be a late event. The mutation-specific signal gradually equaled the wild-type signal, indicating that ras oncogene activation finally affected the great majority of hematopoietic cells. The present study shows that the occurrence of N-ras mutations in MDS may represent an acquired phenomenon in some patients during the course of the disease. In two patients the occurrence of an N-ras mutation was associated with rapidly progressive disease. However, the third patient had a protracted course of the disease over many years after the mutation had been detected, indicating that other factors, which are at present unknown, may contribute to disease progression. ACKNOWLEDGMENT

The authors thank Anneke Kooreman for her help in preparing the manuscript. We acknowledge Dr H.L. Haak for his continuing interest and support.

REFERENCES 1. Koeffler H P Myelodysplastic syndromes (preleukemia). Semin McCormick F, Jacobs A rus mutations in myelodysplasia detected Hematol23:284,1986 by amplification, oligonucleotide hybridization, and transformation. Leukemia 2503,1988 2. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton 13. Ahuja HG, Foti A, Bar-Eli M, Cline MJ: The pattern of DAG, Gralnick HR, Sultan C The FAB Cooperative Group: mutational involvement of rus genes in human hematologic maligProposals for the classification of myelodysplastic syndromes. Br J nancies determined by DNA amplification and direct sequencing. Haematol51:189,1982 Blood 75:1684,1990 3. Prchal JT, Throckmorton DW, Carroll AJ, Fuson EW, Gams 14. Kerkhofs H, Hermans J, Haak HL, Leeksma CHW: Utility RA, Prchal J F A common progenitor for human myeloid and of the FAB classification for myelodysplastic syndromes: investigalymphoid cells. Nature 274590,1978 tion of prognostic factors in 237 cases. Br J Haematol65:73,1987 4. Van Kamp H, Landegent JE, De Graaff E, Willemze R, Fibbe 15. Verlaan-de Vries M, Bogaard ME, Van den Elst H, Van WE: Clonality of hematopoiesis in patients with aplastic anemia Boom JH, Van der Eb AJ, Bos J L A dot-blot screening procedure and myelodysplastic syndromes. Blood 76:50a, 1990 (abstr, suppl) for mutated rus oncogenes using synthetic oligo-deoxynucleotides. 5. Janssen JWG, Buschle M, Layton M, Drexler HG, Lyons J, Gene 50313,1986 Van den Berghe H, Heimpel H, Kubanek B, Kleihauer E, Mufti 16. Melani C, Haliassos A, Chomel JC, Miglino M, Ferraris AM, GJ, Bartram CR: Clonal analysis of myelodysplastic syndromes: Gaetani GF, Kaplan JC, Kitzis A: rus activation in myelodysplastic Evidence of multipotent stem cell origin. Blood 73:248,1989 syndromes: Clinical and molecular study of the chronic phase of 6. Raskind WH, Tirumali N, Jacobson R, Singer J, Fialkow PJ: the disease. Br J Haematol74:408,1990 Evidence of a multistep pathogenesis of a myelodysplastic syn17. Yunis JJ, Boot AJM, Mayer MG, Bos J L Mechanisms of rus drome. Blood 63:1318,1984 mutation in myelodysplastic syndrome. Oncogene 4:609,1989 7. Barbacid M: rus genes. Annu Rev Biochem 56:779,1978 18. Bartram CR, Ludwig W-D, Hiddeman W, Lyons J, Buschle 8. Bos J L rus oncogenes in human cancer: A review. Cancer Res M, Ritter J, Harbott J, Frohlich, Janssen JWG: Acute myeloid 49:4682,1989 leukemia: Analysis of rus gene mutations and clonality defined by 9. Bos JL, Verlaan-de Vries M, Van der Eb AJ, Janssen JWG, polymorphic X-linked loci. Leukemia 3:247,1989 Delwel R, Liiwenberg B, Colly LP: Mutations in N-rus predomi19. Liu E, Hjelle B, Morgan R, Hecht F, Bishop M: Mutations of nate in acute myeloid leukemia. Blood 69:1237,1987 the Kirsten-rus proto-oncogene in human preleukaemia. Nature 10. Janssen JWG, Steenvoorden ACM, Lyons J, Anger B, 300:186,1987 Bohlke JU, Bos JL, Selger H, Bartram CR: rus gene mutations in 20. Hirai H, Kobayashi Y, Mano H, Hagiwara K, Maru Y, acute and chronic myelocytic leukemias, chronic myeloproliferaOmine M, Mizoguchi H, Nishida J, Takaku F A point mutation at tive disorders, and myelodysplastic syndromes. Proc Natl Acad Sci codon 13 of the N-ras oncogene in myelodysplastic syndrome. USA 84:9228,1987 Nature 327:430,1987 11. Lyons J, Janssen JWG, Bartram CR, Layton M, Mufti GJ: 21. Hirai H, Okada M, Mizoguchi H, Mano H, Kobayashi Y, Mutation of Ki-rus and N-rus oncogenes in myelodysplastic synNishida J, Takaku F 'Relationship between an activated N-rus dromes. Blood 71:1707,1988 oncogene and chromosomal abnormality during leukemic progres12. Padua RA, Carter G, Hughes D, Gow J, Farr C, Oscier D, sion from myelodysplastic syndrome. Blood 71:256,1988