Melodysplastic syndromes arising in patients with germline TP53 mutation and Li-Fraumeni syndrome.
The TP53 tumor suppressor gene encodes a transcription factor that induces expression of a wide variety of target genes in response to cellular stress. These target genes regulate critical processes, such as apoptosis, cell cycle arrest, DNA repair, senescence, and cellular metabolism. (11) Unlike mutations in other tumor suppressor genes, which are most often characterized by nonsense mutations that result in a truncated or null protein, most TP53 mutations are missense mutations that result in amino acid substitutions. These abnormal proteins have a decreased ability to bind to DNA and transactivate p53-responsive genes. (12) Most patients with LFS carry a germline mutation in one TP53 allele; in many cases, somatic mutation of the other allele is present in the tumor. Of the small proportion of patients with LFS who present with leukemia, most are affected as children or young adults. The most commonly reported leukemia is acute lymphoblastic leukemia/lymphoma, but acute myeloid leukemia (AML) and chronic myeloid leukemia also occur. (5,13) However, reports of myelodysplastic syndrome (MDS) or AML as second malignancies in patients with germline TP53 mutations or LFS are rare. (14-16) In this study, we present the morphologic, cytogenetic, and molecular diagnostic findings of 3 unique cases of MDS arising in patients with germline TP53 mutation, 2 with classic LFS.
MATERIALS AND METHODS
We searched the Li-Fraumeni Syndrome database of the Department of Genetics at the University of Texas M. D. Anderson Cancer Center (Houston, Texas) and identified 3 patients with documented germline TP53 mutations or LFS who had developed MDS during a period of 6 years (2000-2005). Clinical, cytogenetic, and molecular diagnostic data were available for all 3 patients. Bone marrow core biopsy and aspirate clot sections and aspirate smears were reviewed. The cases were classified according to the World Health Organization classification system. (17) Immunohistochemical staining with antibody to p53 was performed on formalin-fixed, paraffin-embedded tissue sections using the DO-7 antibody (1:50; DAKO, Carpinteria, California), as described previously. (18) This antibody detects both wild type and mutant p53 protein. Staining for p53 was considered positive if at least 10% of the neoplastic cells exhibited unequivocal nuclear staining.
Conventional cytogenetic analysis was performed on metaphases obtained from bone marrow aspirate cultures using standard techniques. Results were reported using the International System for Human Cytogenetic Nomenclature. In one case (case 2), fluorescence in situ hybridization was performed on metaphases using a whole chromosome paint to chromosome 5 (Cytocell Technologies, Ltd., Cambridge, United Kingdom), according to the manufacturer's instructions.
Analysis of the TP53 gene for mutations was performed on DNA obtained from peripheral blood lymphocytes (cases 1 and 3), fibroblasts (case 2), and saliva (case 3). For cases 2 and 3, sequence analysis was performed on all coding exons and flanking splice regions. (19) For case 1, the analysis was restricted to the known familial mutation.
A 47-year-old woman was ascertained as part of a genetic epidemiologic research study of patients with osteosarcoma conducted at our institution in the 1980s, which included her brother as a proband. He had been found to carry a p53 germline mutation. She had a history of multiple malignant neoplasms. Genetic testing for the familial TP53 mutation was performed in 1997 on peripheral blood lymphocytes. Her past medical history was significant for infiltrating ductal carcinoma of the left breast in 1986, for which she underwent a modified radical mastectomy; axillary lymph nodes were negative for metastatic carcinoma. In December 1997, she was diagnosed with glioblastoma multiforme in the right frontoparietal lobe, treated with excision and radiation therapy, followed by 10 months of chemotherapy (procarbazine, lomustine, and vincristine), which was completed in February 1999. Later that year, she developed infiltrating ductal carcinoma of the right breast, which was treated with a modified radical mastectomy. Her family history was remarkable for malignant neoplasms in multiple first-degree relatives, including a history of bladder carcinoma in her mother and osteosarcoma in her brother. Results from a complete blood cell count performed in 1998 were within normal limits. However, during the next 18 months, her peripheral blood cell counts dropped gradually. A complete blood cell count performed in 2000 revealed pancytopenia: white blood cell count of 700/[micro]L (reference range, 4-11/[micro]L), hemoglobin at 8.7 g/dL (reference range, 14-18 g/dL), hematocrit at 28.9% (reference range, 40%-54%), and platelets of 30 X [10.sup.3]/[micro]L (reference range, 140-440 X [10.sup.3]/[micro]L). Examination of a bone marrow core biopsy specimen and aspirate smears, including cytogenetic analysis, confirmed the diagnosis of MDS. Despite treatment with chemotherapy for MDS, the pancytopenia persisted, as did the glioblastoma multiforme. The patient succumbed to recurrent infections 6 months after the diagnosis of MDS.
This patient's case has been reported on previously. (2) She had been seen at our institution since she was 18 months old. At that time, the patient was diagnosed with embryonal rhabdomyosarcoma and was treated with radiation therapy. In 1972, at age 14 years, she developed osteosarcoma of the right mandible, in the previously irradiated area, which was treated with chemotherapy (doxorubicin, vincristine, and cyclophosphamide, an alkylating agent). That was followed by bilateral, infiltrating ductal carcinomas of the right breast in 1989, which was treated with modified radical mastectomy and chemotherapy (fluorouracil, methotrexate, and vincristine), and in the left breast in 1992, treated only with modified radical mastectomy. In 2001, at age 44 years, she presented with a soft tissue mass of the right maxilla. A biopsy specimen obtained from the right maxillary mass demonstrated osteosarcoma. She underwent excision and orofacial reconstruction, followed by 6 cycles of chemotherapy with ifosfamide. The patient and family members had extensive histories of malignant neoplasms. Both of her brothers died from cancer, one at age 24 years from maxillary osteosarcoma, the other, at age 27 years from an adenocarcinoma of the gastrointestinal tract following osteosarcoma and acute lymphoblastic leukemia/lymphoma. Her mother had died at age 32 from metastatic breast carcinoma. Given the extensive personal and family history of cancer, samples of fibroblasts and peripheral blood lymphocytes obtained in 1979 and 1982, respectively, had been assessed for TP53 mutation. (2) Results from a complete blood cell count, performed in 2002, following chemotherapy for the osteosarcoma, were within reference range. However, during a routine clinic visit in August 2005, the patient was found to have pancytopenia. Examination of a bone marrow core biopsy specimen and aspirate smears, including cytogenetic analysis, revealed MDS, which evolved to AML during the next 6 months. She underwent allogeneic stem cell transplantation in August 2006 for persistent AML. Her posttransplant course was complicated by graft-versus-host disease and recurrent infections. She succumbed to pneumonia 10 months after the diagnosis of MDS.
A 7-year-old boy was referred to the Cancer Genetics Clinic in June 2005 with a history of 3 primary cancer diagnoses. Peripheral blood lymphocytes and saliva obtained from the patient and peripheral blood lymphocytes obtained from his parents were assessed for TP53 mutation. In 1998, at age 11 months, he was diagnosed with embryonal rhabdomyosarcoma of the right maxilla and underwent partial maxillectomy and temporal bone resection, followed by chemotherapy (vinblastine) and radiation therapy. In 2003, at age 5 years, he developed osteosarcoma of the right mastoid and was treated with mastoidectomy and right neck dissection, followed by chemotherapy (agent unknown) and radiation therapy. After completing treatment in 2004, test results showed anemia (hemoglobin, 9.4 g/dL) and thrombocytopenia (platelets, 63 X [10.sup.3]/[micro]L), with a normal white blood cell count (6400/[micro]L). During the next 12 months, bone marrow core biopsies and aspirate smears showed hypercellularity with left-shifted myeloid maturation and dysplastic features and with between 8% and 15% blasts. In September 2005, repeat bone marrow sampling that included cytogenetic analysis demonstrated evolution to AML. The patient was the only child, and there was no history of malignant neoplasms in either parent. The patient underwent allogeneic bone marrow transplantation but was subsequently lost to follow-up.
The clinical and molecular diagnostic findings are summarized in Table 1. Two patients were female, and one was male. Two patients (cases 2 and 3) presented with malignant neoplasms in early childhood; one patient (case 1) presented as an adult. All patients developed at least 2 LFS-associated malignant neoplasms. Two patients had embryonal rhabdomyosarcoma and osteosarcoma as children, with the osteosarcomas arising in previously irradiated areas (cases 2 and 3); 2 had bilateral breast carcinoma (cases 1 and 2), and one had glioblastoma multiforme (case 1). All patients received treatment with chemotherapy and radiation therapy.
All patients developed MDS (Figure 1, A and B), which evolved to AML in 2 patients (cases 2 and 3). The morphologic features in the bone marrow specimens and the results of the cytogenetic analyses are summarized in Table 2. Conventional cytogenetic analyses performed on bone marrow cultures demonstrated complex karyotypes with abnormalities of chromosome 5 in all cases; 2 cases also had monosomy 7 (cases 1 and 2; Table 2; Figure 2, A and B). In one case (case 3), the chromosome abnormality der(5)t(5;17)(p10;q10) resulted in loss of one of the long arms of chromosome 5, as well as loss of one of the short arms of chromosomes 17, the site of the TP53 gene. Chromosome 17 in the other 2 cases showed no abnormalities on conventional cytogenetic analysis.
Immunohistochemical staining for p53 was performed in the 2 cases for which tissue sections were available. In case 2, results from stains performed on both a bone marrow core biopsy involved by MDS and the osteosarcoma specimen were negative (data not shown). In contrast, in case 3, results from the stain performed on a bone marrow core biopsy involved by AML was strongly positive in most of the nuclei of the marrow elements (Figure 3, A and B).
Germline mutation of the TP53 gene was identified in all patients (Table 1). Two patients (cases 1 and 2) had family histories that were significant for malignant neoplasms in multiple first-degree relatives and, therefore, met the criteria for classic LFS. In case 3, the family history was negative, and neither parent demonstrated the mutant allele. In case 1, sequence analysis of the TP53 gene, restricted to the known familial mutation, was performed, 3 years before the patient developed MDS, on DNA that was obtained from peripheral blood lymphocytes. This study demonstrated a missense mutation at codon 273 in exon 8 (CGT>CAT), resulting in an amino acid change from arginine to histidine. In cases 2 and 3, sequence analysis of all coding exons and flanking splice regions was performed. In case 2, analysis of DNA obtained from skin fibroblasts and peripheral blood lymphocytes more than 20 years before the diagnosis of MDS demonstrated a single base deletion in exon 5 that ultimately led to a stop codon. In case 3, sequence analysis performed on DNA obtained from peripheral blood lymphocytes and saliva at the time of the diagnosis of MDS demonstrated a missense mutation at codon 241 in exon 7 (TCC>TTC), resulting in an amino acid change from serine to phenylalanine.
The human TP53 gene encompasses 20 kb of DNA, consisting of 11 exons; the first noncoding exon separated from the cluster of 10 exons by a large intron of 10 kb. (20) Most TP53 mutations in human cancers are missense mutations that result from single-nucleotide substitutions. (20) Although TP53 mutations can occur anywhere in the gene, most substitutions are found within "hot spots" in the DNA-binding domain of the protein, in exons 5 through 8. About 15% of somatic mutations are found outside of this region. (20) Frameshift or nonsense mutations are less common. The germline mutations in all 3 patients were found within exons 5, 7, and 8. The mutation in case 1, one of the most common TP53 mutations in both sporadic and LFS-associated cancers, is predicted to result in a nonfunctional protein. (21) The germline mutation in case 2 is a frameshift mutation that ultimately results in a stop codon and would not be expected to yield a protein. The germline mutation in patient 3 is an uncommon missense mutation that, similar to case 1, is also predicted to result in a nonfunctional protein. (21) Unlike cases 1 and 2, case 3 had no family history of malignant neoplasms and, therefore, did not meet the criteria for classic LFS. Further, DNA sequence analysis failed to demonstrate TP53 mutation in either parent. Thus, it seems likely that the multiple primary cancers in this patient arose in the setting of a new germline TP53 mutation.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Immunohistochemical staining for p53 protein in the 2 cases tested was concordant with the results of the TP53 sequence analysis. Case 3, with a missense mutation, showed strong nuclear positivity in the AML cells in the bone marrow. In contrast, in case 2, with a nonsense mutation, neither the osteosarcoma specimen nor the bone marrow specimen involved by AML were positive for p53. However, factors other than mutation status contribute to variability in staining with antibody to p53. (22) These include different p53 isoforms or posttranslational modifications that may not be recognized by a particular antibody. Technical factors, such as the quality of the fixation, effects of antigen retrieval, antibody concentration, and others, may also affect staining. Thus, conclusions about the mutation status of TP53 based on the results of immunohistochemical staining for the protein should be made with caution.
Several large series have demonstrated that cytogenetics in patients with MDS have a major effect on the survival and evolution to AML. One of the largest studies, which led to the International Prognostic Scoring System for MDS, found that patients with MDS could be divided into 3 prognostic subgroups based on the cytogenetic findings. (23) In this study, cytogenetic findings associated with a "good" outcome were a normal karyotype, --Y alone, del(5q) alone, or del(20q) alone; the median survival for this subgroup of patients was 3.8 years. Cytogenetic findings associated with a "poor" outcome were a complex karyotype ([greater than or equal to] 3 abnormalities) or abnormalities of chromosome 7; the median survival in this group was 0.8 years. All other findings of cytogenetic abnormalities were associated with an "intermediate" outcome and a median survival of 2.4 years. Based on the cytogenetics findings, all 3 patients in our study would be considered to be in the "poor" outcome group; all had abnormalities of chromosome 5 as part of a complex karyotype with 3 or more abnormalities, and 2 also showed monosomy 7.
[FIGURE 3 OMITTED]
Mutations in TP53 are relatively uncommon in cases of primary (de novo) MDS, occurring in fewer than 25% of cases. (24-26) In these studies, the mutation status was evaluated in DNA obtained from involved bone marrow. Thus, it is not clear whether the mutations represent germline or somatic mutations. Regardless, when mutations occur, they are usually found in the high-risk morphologic subtypes of MDS that are associated with a poor outcome and a propensity to evolve to AML, that is, refractory anemia with excess blasts (RAEB) and refractory anemia with excess blasts in transformation (RAEBT). They are unusual in the morphologically low-risk subtypes of MDS, that is, refractory anemia or refractory anemia with ringed sideroblasts. Wattel and coworkers (24) reported that the neoplastic cells in 19 of 88 patients (22%) with RAEB or RAEB-T harbored a TP53 mutation, compared with only 1 out of 94 patients (1%) with other MDS subtypes. Similarly, Sugimoto and coworkers (27) found that 11 out of 50 patients (22%) with RAEB or RAEB-T had TP53 mutations, but only 5 out of 68 patients (7%) with other MDS subtypes had a TP53 mutation. Mutations in TP53 are associated with a complex karyotype, which often includes deletions in the chromosome 17 short arm. (26) Patients with MDS and TP53 mutations have a poor overall survival rate and are at increased risk for progression to AML.
It seems likely that the MDS that arose in our patients were therapy related. Patients who have received a wide variety of chemotherapeutic agents or radiation therapy are at increased risk to develop therapy-related MDS (t-MDS) and AML (t-AML). Particular classes of chemotherapeutic agents are associated with specific types of cytogenetic abnormalities. (26) Patients treated with topoisomerase II inhibitors tend to present with overt AML characterized by balanced translocations or inversions after a latency period of 1 to 5 years. Patients treated with alkylating agents and/or ionizing radiation tend to present with t-MDS characterized by deletions or loss of chromosomes 5 and 7 after a latency period of 5 to 10 years. In addition, there is a relatively high incidence of point mutations in TP53 in patients with t-MDS/t-AML compared with primary MDS and AML. In a series of 140 patients with t-MDS/t-AML, Pedersen-Bjergaard and coworkers (28) found TP53 point mutations in 34 cases (24%), often associated with loss of heterozygosity. In our study, all 3 patients had received at least one course of chemotherapy with an alkylating agent before developing MDS, and all received at least 2 courses of radiation therapy. Ionizing radiation is known to increase the risk of malignant neoplasms in patients with LFS, particularly within the radiation field. (29) Given the rarity of MDS in LFS patients, the occurrence of radiation-related osteosarcoma in 2 of the 3 patients with MDS (67%) suggests a unique susceptibility to DNA-damaging agents. Although we do not know the mutation status of their second TP53 alleles, it is likely that exposure to both chemotherapy and radiation therapy in the setting of germline TP53 mutation predisposed these patients to develop t-MDS/t-AML.
In conclusion, we report 3 unique cases of MDS arising in patients with germline TP53 mutations, 2 with classic LFS. Although rare compared with solid tumors, patients with LFS may develop MDS, which is most likely therapy related and is associated with cytogenetic markers of poor prognosis.
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Lynne V. Abruzzo, MD, PhD
Accepted for publication September 29, 2009.
From the Departments of Hematopathology (Drs Talwalkar, Yin, and Abruzzo) and Cancer Genetics (Dr Strong), University of Texas M. D. Anderson Cancer Center, Houston, Texas; and the Departments of Pediatrics (Dr Naeem) and Pathology (Dr Hicks), Baylor College of Medicine, Houston.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Lynne V. Abruzzo, MD, PhD, Department of Hematopathology, University of Texas M. D. Anderson Cancer Center, Box 350, 1515 Holcombe Blvd, Houston, TX 77030.
Table 1. Summary of Clinical and Molecular Diagnostic Findings Case Age/ Germline TP53 Previous Malignant No. Sex Mutation Neoplasms 1 33 y/F Missense, exon 8, Bilateral breast CGT.CAT, p.R273H, carcinoma, (Arg.His) glioblastoma multiforme 2 18 mo/F Frameshift, single base Embryonal deletion exon 5, rhabdomyosarcoma, 184delT bilateral breast carcinoma, osteosarcoma 3 11 mo/ Missense, exon 7, Embryonal M TCOTTC, p.S241F, rhabdomyosarcoma, (Ser.Phe) osteosarcoma Age at Case Latency, (a) t-MDS No. Previous Therapy y Diagnosis, y 1 Radiation therapy, 1.5 47 procarbazine, lomustine, vincristine 2 Radiation therapy, 46 48 doxorubicin, vincristine, cyclophosphamide, fluorouracil, methotrexate, vincristine, ifosfamide 3 Vinblastine, radiation 6.5 ~7.5 therapy Outcome/Time Case After t-MDS No. Diagnosis, mo 1 Died of GBM/6 2 Died of t-AML/ 10 3 Developed t- AML/1; lost to follow-up Abbreviations: GBM, glioblastoma multiforme;t-AML, therapy-related acute myeloid leukemia;t-MDS, therapy-related myelodysplastic syndrome. (a) Latency is the period between initiation of the first treatment with either radiation therapy or chemotherapy and development of t-MDS. Table 2. Summary of Bone Marrow Morphology and Conventional Cytogenetic Findings Case Cellularity, Myeloblasts, No. % % 1 40 2 2 30 3 3 95 33 Case No. Dysplasia Findings Diagnosis 1 RBC: megaloblastoid, 65% RS; t-MDS Meg: small, hypolobated 2 RBC: nuclear irregularity and t-MDS karyorrhexis, cytoplasmic vacuolization, 8% RS; Myeloid: nuclear atypia, cytoplasmic hypogranularity and vacuolization, pseudo-Pelger-Huet nuclei; Meg: small, hypolobated, clustered 3 RBC: megaloblastoid, nuclear budding, t-AML arising cytoplasmic vacuoles, 52% from t-MDS nucleated RBC precursors Myeloid: blasts with rare granules, no Auer rods Meg: small, hypolobated, clustered Case No. Conventional Cytogenetic Results 1 45,XX,del(5)(q15q33), - 7/ 45,idem,del(12)(p11.2)/ 46,idem,+8,del(12)(p11.2)/46,XX 2 46,XX,r(5),t(5;13)(q13;q12), - 7,+mar 3 44,XY,-4,der(5)t(5;17)(p10;q10),-20, add(22)(q13),+mar[cp7]/ 4 clonally related sidelines Abbreviations: Meg, megakaryocyte;RBC, red blood cells; RS, ringed sideroblasts;t-AML, therapy-related acute myeloid leukemia; t-MDS, therapy-related myelodysplastic syndrome.