Analysis of P73 and P53 Gene Deletions in Multiple Myeloma B Schultheis1, a Kra¨Mer1, a Willer1, U Hegenbart2, H Goldschmidt2 and R Hehlmann1

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Leukemia (1999) 13, 2099–2103  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Analysis of p73 and p53 gene deletions in multiple myeloma B Schultheis1, A Kra¨mer1, A Willer1, U Hegenbart2, H Goldschmidt2 and R Hehlmann1

1III Medizinische Klinik, Klinikum Mannheim, Universita¨t Heidelberg, Mannheim; and 2Medizinische Klinik und Poliklinik V, Universita¨t Heidelberg, Heidelberg, Germany

Recently, p73, a protein with structural and functional simi- ing various splicing variants have been identified as putative larities to p53, an extensively studied tumor suppressor gene, p53 family members.6–12 Mutations or deletions of genes has been cloned. After being mapped to the chromosomal region 1p35–1p36, it has been postulated to act as a tumor sup- encoding these proteins have rarely been found in human 8,13–21 pressor gene, too, as this region is altered in several human cancers or hematological malignancies so far. malignancies. Deletions of the short arm of chromosome 1 After being mapped to the chromosomal region 1p35–1p36, have frequently been described in multiple myeloma (MM) p73 has been postulated to act as a tumor suppressor gene, whereas structural abnormalities of the 17p13 region including as this region was shown to be deleted in several human p53 are rare events in this disease. Since it has been proposed malignancies including neuroblastoma, pheochromocytoma, that especially neoplasias lacking p53 alterations might show a loss of heterozygosity at 1p35–1p36, we studied the fre- oligodendroglioma, melanoma, merkel cell cancer, germ cell quency of p53 and p73 deletions in bone marrow mononuclear cancer, breast cancer, ovarian cancer, liver cancer and colon cells of 68 patients with MM, two patients with monoclonal gam- cancer as well as in some precancerous lesions like colorectal mopathy of undetermined significance and four patients with adenomas.20,22–26 Recently published preliminary data dis- plasma cell leukemia. Dual-color fluorescence in situ hybridiz- cussed the role of p73 in hematologic neoplasias,27,28 as well ation (FISH) for p53 and p73 was performed using commercially 29 23 available DNA probes for 17p13.3 and the microsatellite marker as in renal cell carcinoma and lung cancer. Although D1Z2, respectively. Centromeric DNA probes served to dis- initial analysis of neuroblastoma cell lines failed to reveal p73 tinguish gene deletions from whole chromosome losses. In mutations, Kaghad et al6 suggested that monoallelic transcrip- contrast to recently published FISH results, we only detected tion of p73 and absence of protein expression in neuroblas- heterozygous p53 deletions in eight out of the 74 patients, three tomas with hemizygous 1p36 deletions were consistent with of them showing a monosomy 17. Heterozygous deletions of the D1Z2 region at 1p36 were found in six cases with one involvement in tumor development supporting the notion that patient having a monosomy 1. Neither homozygous deletions p73 might act as a tumor suppressor gene in malignancies of either chromosomal region nor nullisomies 1 or 17 could with 1p36 deletions. However, the chromosomal region 1p36 be detected. These results argue against a major role of p73 is thought to harbor at least two tumor-suppressor genes.30 deletions in MM. As MM patients with 1p structural abnormali- p73 has been mapped to the distal border of the minimal con- ties have a significantly poorer survival rate than those with sensus region of neuroblastoma, 1p36.2–3, limited by the normal karyotypes, the role of other putative tumor suppressor 31 genes located at the chromosomal region 1p36 in the patho- microsatellites D1Z2 and D1S47. genesis of MM has to be determined. Multiple myeloma (MM) is a B cell neoplasia characterized Keywords: p53; p73; multiple myeloma; FISH; gene deletions by the proliferation of a malignant plasma cell population. Due to a low percentage of plasma cell mitoses in comparison to normal hematopoietic progenitor cells, numeric or struc- Introduction tural chromosomal abnormalities could only be detected in 30–50% of patients by conventional cytogenetic studies,32–34 The tumor suppressor gene p53, located at the chromosomal whereas flow cytometry,35 comparative genomic hybridiz- region 17p13, is known to be deleted in a variety of human ation,36 measurement of DNA content37,38 and interphase neoplasms.1 p53 has been implicated in the control of cellular fluorescence in situ hybridization (FISH)35 revealed an up to proliferation, differentiation and apoptosis, and its ability to 90% incidence of aneuploidy in MM patients.36,39,40 Among inhibit cell growth is due, at least in part, to transcriptional the structural chromosomal aberrations found, abnormalities activation of target genes such as that encoding the cell cycle of chromosome 1p are known to occur frequently in MM WAF1/Cip1 41,42 inhibitor p21 leading to G1/S-phase arrest and patients whereas structural changes of the short arm of apoptosis. In hematological malignancies, p53 mutations chromosome 17 have been described to a much smaller occur at a lower frequency compared with solid tumors, but extent. Also, in this malignancy p53 mutations are very rare when present they appear to be prognostically relevant in at the time of diagnosis and even in patients with advanced patients with aggressive or advanced stages of disease, as it or aggressive forms of MM, p53 mutations were found in no has been found in non-Hodgkin’s lymphomas, myelodysplas- more than 20% of patients.43 Since it has been proposed that tic syndromes, acute myeloid leukemia and blastic crisis of especially neoplasias lacking p53 alterations might show a 2–5 chronic myelogenous leukemia. loss of heterozygosity at 1p35–1p36,6 we studied the fre- Recently, several proteins showing functional or structural quency of deletions of p53 and its proposed new homolog similarities to p53 have been described, suggesting that p53 p73 at 1p36 in bone marrow mononuclear cells of MM belongs to a gene family whose function still remains to be patients. Additionally, since various structural and numerical exactly defined. Several isoforms of the human homolog of chromosome aberrations in MM like partial or complete the rat Ket gene, named p51, p63, p73, p73L and p40, includ- deletions of chromosome 13 and 11q abnormalities44 or, as recently concluded, p53 deletions45 are predictive for a worse prognosis, and since chromosomal abnormalities of 1p have Correspondence: Dr B Schultheis, at her present address: Department of Haematology, ICSM, Hammersmith Hospital, Ducane Road, been found to be associated with shorter event-free and over- 44 London W12 0NN, UK; Fax: 44 181 742 9335 all survival, we sought to investigate if possible 1p36 Received 29 June 1999; accepted 30 August 1999 deletions were correlated with advanced disease. p53 and p73 deletions in multiple myeloma B Schultheis et al 2100 Materials and methods Results

Patients Clinical features

Bone marrow samples from 68 MM and two MGUS patients Heparinized bone marrow samples as well as bone marrow as well as blood samples from four patients with PCL were smears were obtained from 68 patients with MM and two with analyzed. The median age of the patients was 56 years (range monoclonal gammopathy of undetermined significance 36 to 77 years). According to the staging system of Durie and (MGUS). Peripheral blood samples from four patients with Salmon,48 54 out of the 68 MM patients had stage III myeloma plasma cell leukemia (PCL) were also included. Bone marrow whereas only five and nine patients could be classified as suf- and peripheral blood samples were taken after informed con- fering from stage I or stage II myeloma, respectively. Female sent according to the Declaration of Helsinki. Peripheral and male patients were equally distributed among the groups. blood samples from eight normal individuals served as M-protein gradients revealed a percentage of 62% (46/74) controls for determination of cut-off values. IgG, 18% (13/74) IgA, 16% (12/74) light-chains only, 1% (1/74) IgD and 3% (2/74) nonsecretory myeloma, MGUS, or PCL, thus reflecting currently known distributions. Sixteen out Determination of plasma cell content of the 74 patients were studied at initial diagnosis whereas 58 patients had been receiving chemotherapy and/or radio- Plasma cell contents of the bone marrow and peripheral blood therapy before. Median time from diagnosis was 1.5 years samples were estimated either by conventional May–Gru¨n- (0.0–22.8 years) for all patients vs 2.0 (0.2–22.8) years for wald-Giemsa staining or by flow cytometry after staining with patients under therapy (Table 1). CD38 and CD138 antibodies using the CycloScope kit (Medac, Hamburg, Germany) according to the manufacturer’s instructions. Structural and numerical chromosomal abnormalities of chromosomes 1 and 17 as detected by FISH

Interphase FISH revealed a monoallelic deletion of the 1p36 Fluorescence in situ hybridization (FISH) region in six of 61 evaluable patients (9.8%), one of them showing a monosomy 1. No homozygous 1p36 deletions Heparinized bone marrow samples or peripheral blood could be detected. Eight out of 61 patients (13.1%) had a samples were subjected to Ficoll gradient centrifugation monoallelic p53 deletion, three of them with a monosomy 17 (density 1.077 g/ml, Pharmacia, Freiburg, Germany). Buffy coats were washed once with phosphate-buffered saline (PBS). Table 1 Patient characteristics Part of the specimen was used for morphological analysis of May–Gru¨nwald-Giemsa-stained slides or flow cytometry; the Feature No. of patients remainder of the cell suspension was brought on to slides, − ° fixed in methanol/acetic acid 3:1 and stored at 70 C. Dual- Total 74 color FISH was performed following standard protocols with Male 38 (51%) slight modifications.46 Briefly, slides were fixed in methanol Female 36 (49%) for 10 min, digested in 0.01 N HCl containing 3–5 mg/100 ml Age 56 pepsin (Serva, Heidelberg, Germany), washed twice in PBS (median, range 36–77) Diagnosis and fixed with 1% paraformaldehyde (Sigma, Deisenhofen, MGUS 2 Germany) and 5 mM MgCl2 (Merck, Darmstadt, Germany) in MM 68 PBS. After dehydration in 70%, 90% and 100% ethanol for Stagea 10 min each, slides were denatured in 70% formamide I5 (Merck) in 2 × SSC for 2 min at 72°C. After incubation in II 9 III 54 100 mM ice-cold CaCl and a second dehydration step, 2 Bone marrow plasma cells hybridization was performed for 12 to 16 h. Commercially Ͻ10% 11 available DNA probes for p53 (Vysis, Stuttgart, Germany) and 10–30% 25 p73/1p36.3 (D1Z2) (Oncor, Heidelberg, Germany) were used у30% 32 with centromeric DNA probes for chromosomes 17 D17Z1 PCL 4 (Vysis) and 1 (D1Z5) (Oncor) as controls.47 Hybridization with M-protein Oncor probes was performed as described previously. Vysis IgG 46 IgA 13 DNA probes were treated following the instructions of the IgD 1 manufacturer. Slides were counterstained in Vectashield Bence–Jones only 12 (Vector, Burlingame, CA, USA) anti-fading medium containing nonsecretory 2 4,6-diamino-2-phenylindole-dihydrochloride (DAPI) 1 ␮g/ml. Treatment Evaluation was performed by analyzing at least 200 interphase No prior treatment 16 nuclei per sample using fluorescence microscopy. Means and Previous chemotherapy 58 Time from diagnosis standard deviations (s.d.) of the percentages of nuclei with 0, All patients 1.5 years 1, and 2 domain signals were calculated for the control speci- (median, range 0.0–22.8) mens. A cut-off value for a decision of mono- or nullisomy Patients under treatment 2.0 years and deletions was based on the percentage of cells with zero (median, range 0.2–22.8) and one signal, respectively, being greater than the mean percentage ± 3 s.d. of the controls. aStage according to the classification of Durie and Salmon.48 p53 and p73 deletions in multiple myeloma B Schultheis et al 2101 Table 2 Patients with p53 and/or p73 deletions

Pat. no. Stagea Plasma cell content time after Dx (years) 1p36 del centr 1 del 17p del centr 17 del in the bone marrow

1 III 10% 3.2 10.8% NS NS NS 2 III 20–30% 0.4 10.8% NS NS NS 3 III 10–20% 8.0 10.6% NS NS NS 4 II 20–40% 0.4 NS NS 12.2% NS 5 III Ͼ95% 2.0 9.6% NS NS NS 6 III 50% 2.4 12.8% 10.9% NS NS 7 III 80–100% 1.9 19.0% NS 14.6% NS 8 III 50–100% 0.4 NS NS 31.0% NS 9 III Ͼ95% initial diagnosis NS NS 11.5% 5.3% 10 III 50% 6.5 NS NS 34.2% 34.2% 11 III 10–30% 7.1 NS NS 13.6% NS 12 PCL 50% initial diagnosis NS NS 33.8% 6.5% 13 PCL 50% initial diagnosis NS NS 11.6% NS

cut-off (mean ± 3 × standard deviation, n = 8) 9.5% 5.8% 10.6% 4.4% aStage according to the classification of Durie and Salmon.48 NS, not significantly elevated. in a significant number of cells. Only one of the patients had p53 family members being described, has been mapped to both, a 1p36 as well as a p53 deletion (Table 2). Cut-off 1p36, a region frequently altered in solid tumors and hemato- values, determined by analysis of peripheral blood samples logical neoplasias. In MM, up to two thirds of patients show from eight normal individuals for each DNA probe, were structural aberrations of the short arm of chromosome 1,42 9.6% for 1p36 deletions (mean 3.6%, s.d. 2.0%), 5.9% for whereas p53 deletions or mutations have rarely been monosomy 1 (mean 1.4%, s.d. 1.5%), 10.5% for 17p13 found.49,50 Since structural and functional similarities between deletions (mean 4.5%, s.d. 2.0%) and 4.4% for monosomy 17 p53 and p73 have been described, one may hypothesize that (mean 1.7%, s.d. 0.9%), respectively. Therefore, both patients p73 acts as a tumor suppressor gene especially in neoplasias with MGUS and 11 patients with bone marrow plasma cell lacking p53 abnormalities. contents below 10% had to be considered as non-evaluable. In this study, we first present evidence arguing against a Both 1p36 and 17p13 deletions were found almost exclus- major role of p73 as a tumor suppressor gene in MM develop- ively in patients suffering from advanced disease, ie PCL or ment. The importance of p73 alterations in human malig- myeloma stage III (6/6 1p36 deletions vs 7/8 17p13 deletions). nancies has been controversely discussed so far. Several While none of the patients with newly diagnosed MM or PCL groups have analyzed p73 gene alterations as well as the had a deletion of the microsatellite D1Z2 at chromosomal expression status of the p73 protein in various tumor cell lines region 1p36.3, p53 deletions at 17p13 could be detected in and primary cells of different solid tumors. However, consist- 21% (3/14) of patients at initial diagnosis (Table 3). ent with our observations in monoclonal plasma cell disorders, no deletions or mutations of the p73 gene which would support the role of p73 as a tumor suppressor gene Discussion have been reported so far.14,15,18–20,51 Importantly, even in neuroblastomas, recent data on p73 deletion, mutation, and The tumor suppressor gene p53 has recently been suggested to expression argue that p73 is unlikely to be a tumor suppressor belong to a family of structurally and functional related genes gene inactivated during neuroblastoma development.52 including various splicing variants. p73, the first of the new Second, we present data showing that p53 deletions also occur only in a minority of MM patients. As observed with Table 3 p53 and/or p73 deletions in selected subgroups of patients p73 deletions, the presence of p53 deletions was associated with stage III disease. However, while none of the patients No. of patients 1p36 del centr 17p del centr with PCL had a 1p36.3 deletion, two out of four patients with 1 del 17 del PCL had a p53 deletion. These findings are in agreement with other studies, demonstrating that loss of p53 alleles and With initial somatic mutations of p53 exons 5 to 9 have only been found diagnosis 14 0 0 3 2 at a low frequency in MM.5,49,50,53,54 Also, p53 alterations are Under treatment 47 6 1 5 1 rare in other hematological malignancies like low-grade non- MM stage I Hodgkin’s lymphomas or chronic myelogenous leukemia and or IIa 130010 occur predominantly in advanced disease stages.2,4 In con- MM stage III 44 6 1 5 2 trast, a single recent study described p53 deletions in almost PCL 4 0 0 2 1 one third of all MM patients at initial diagnosis and even in 40 Total 61 6 1 8 3 54.5% of patients with relapsed MM as evaluated by FISH. However, since 65.2% of the patients analyzed by Drach et 40 Only patients with a bone marrow plasma content Ͼ10% have al but 73% of the patients in our study had stage III disease, been included. the different frequencies of p53 deletions might be explained aStage according to the classification of Durie and Salmon.48 by the differences in stage or age distribution of patient p53 and p73 deletions in multiple myeloma B Schultheis et al 2102 samples analyzed. In his patient group, 72% of patients were 11 Senoo M, Seki N, Ohira M, Sugano S, Watanabe M, Inuzuka S, older than 60 years in contrast to 27 out of 74 or 26 out of Okamoto T, Tachibana M, Tanaka T, Shinkai Y, Kato H. A second 61 evaluable patients in our study (36 or 43%, respectively). p53-related protein, p73L, with high homology to p73. Biochem Biophys Res Commun 1998; 248: 603–607. Moreover, most of our patient samples were taken during 12 Schmale H, Bamberger C. A novel protein with strong homology treatment, where Drach et al did not find any p53 deletions to the tumor suppressor p53. Oncogene 1997; 15: 1363–1367. in a limited number of samples investigated. 13 Kroiss M, Bosserhoff A, Vogt T, Buettner R, Bogenrieder T, Land- The findings reported here demonstrate for the first time that thaler M, Stolz W. Loss of expression or mutations in the p73 p73 deletions are, analogous to p53 deletions, rare in MM. As tumour suppressor gene are not involved in the pathogenesis of with other neoplastic disorders, these findings argue against a malignant melanomas. Melanoma Res 1998; 8: 504–509. 14 Nimura Y, Mihara M, Ichimiya S, Sakiyama S, Seki N, Ohira M, major role of p73 as a tumor suppressor gene in MM develop- Nomura N, Fujimori M, Adachi W, Amano J, He M, Ping Y, Naka- ment in a classic Knudson’s manner despite known alterations gawara A. p73, a gene related to p53, is not mutated in esophageal of chromosomal region 1p36 in this disorder. However, also carcinomas. Int J Cancer 1998; 78: 437–440. in analogy to other hematological malignancies, rare deletions 15 Stirewalt DL, Clurman B, Appelbaum FR, Willman CL, Radich JP. of p53 as well as p73 seem to be associated with advanced p73 mutations and expression in adult de novo acute myelogen- disease stages in MM. Since some studies have indicated that ous leukemia. Blood 1998; 92: 221a (Abstr.). aberrant p73 gene expression levels might play a role in tumor 16 Yokomizo A, Mai M, Tindall D, Cheng L, Bostwick D, Naito S, 55 Smith D, Liu W. Overexpression of the wild type p73 gene in growth or progression, mutational as well as expression human bladder cancer. 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