p53 protein expression independently predicts outcome in patients with lower-risk myelodysplastic syndromes with del(5q)

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nchemistry (IHC) has been used as a surrogate marker for TP53 gene mutations in hematologic and other malignancies, including large B-cell lymphoma. [22][23][24] Recently, it was demonstrated that p53 nuclear expression correlated with hemizygous TP53 mutation and outcomes in relapsed myeloma patients treated with lenalidomide. 25 AML following MDS has a dismal prognosis, which makes prediction essential, especially in patients who potentially could be cured by stem cell transplantation. In order to develop a clinically useful prognostic tool, we measured p53 protein expression by IHC in a cohort of 85 patients with lower-risk del(5q) MDS from the MDS-004 clinical trial, and compared outcomes and responses to lenalidomide treatment in patients with respect to strong nuclear p53 expression. We demonstrate the independent prognostic value of p53 IHC in this population of patients, and show that strong nuclear staining reflects underlying TP53 mutations. Our findings underscore the importance of including molecular markers such as TP53 mutations in risk-assessment for del(5q) MDS patients. Methods PatientsFormalin-fixed paraffin-embedded BM trephines from patients enrolled in the phase III, randomized, double-blind, placebo-controlled MDS-004 trial were retrieved. 5 The MDS-004 trial assessed the efficacy and safety of lenalidomide in 205 red blood cell transfusion-dependent patients with low-or intermediate-1-risk del(5q) MDS. The inclusion criteria and treatment schedule were as previously described; 5 a bone marrow biopsy was recommended, but not mandatory. The present study was conducted under the ethical consent for the MDS-004 trial. The original ethical permit did not include any type of sequencing; therefore, TP53 deep-sequencing analysis was only possible in a subset of patients who were still alive and provided consent to an ethical permit obtained after the MDS-004 trial had been completed. In Sweden, gene-sequencing analysis was performed under a separate national ethical permit, which was used for the pyrosequencing of laser-microdissected BM cells. Bone marrow morphology and immunohistochemistryOverall, 131 BM trephines from 85 of the 205 patients (41%; IHC study cohort) obtained at baseline and follow-up were assessed in a blinded fashion. Serial BM biopsies were available for 25% (21 of 85) of the patients. BM cellularity and fibrosis were assessed according to European consensus guidelines. 26 The percentage and intensity of p53 staining was assessed based on a total manual count of 1000 BM hematopoietic cells (lymphocytes/lymphoid aggregates excluded) and graded as: 0 (negative); 1+ (weakly positive); 2+ (moderately positive); and 3+ (strongly positive). The entire BM trephine was also assessed using a Modified Quick Score; 27 a score of ≥3 was used to define p53-positive staining as previously described. 28 In addition, all samples were assessed blindly for the percentage of p53-DO1 strongly positive (3+) cells by three hematopathologists fro...

High p53 protein expression in therapy-related myeloid neoplasms is associated with adverse karyotype and poor outcome

“…In patients with low-risk myelodysplastic syndrome with del(5q), it was also shown that the presence of Z1% p53-expressing cells was positively correlated with TP53 mutations. 13,22 We also found an excellent correlation betweenZ1% p53 3 þ cells and the presence of TP53 mutation in the 71 cases subjected to sequencing in our study: 29/33 cases (88%) with TP53 mutation were scored as p53 immunohistochemistry-positive. The four falsenegative cases included two cases with a nonsense mutation truncating the protein, likely explaining the loss of detection by immunohistochemistry.…”

“…4,13,15,21,22 To our knowledge, the current study is the first to p53 Expression in therapy-related myeloid neoplasms examine p53 protein expression in a large series of therapy-related myeloid neoplasms. We scored p53 by manual counting and found that, using a cutoff similar to that used in low-risk myelodysplastic syndrome (Z1% 3 þ cells), 22 positivity for p53 was strongly associated with poor outcome in therapy-related myeloid neoplasms. This adverse association was independent of other factors known to influence prognosis, such as age, acute myeloid leukemia vs myelodysplastic syndrome, karyotype risk group, and treatment with stem-cell transplantation.…”

“…Saft et al 22 showed by single-cell microdissection that cells with weak to moderate p53 staining lack TP53 mutation. Therefore, in counting p53 immunostained cells, it is important to avoid counting moderately or weakly stained cells.…”

“…At least 300 cells (median 610 in test set and 576 in validation set) were counted except in cases with 415% p53 3 þ cells present throughout the biopsy and in one paucicellular case in which no p53 3 þ cells were identified in all 241 mononuclear cells available. On the basis of prior studies, 22 a cutoff of Z1% p53 3 þ cells was used to define positive p53 staining (p53 immunohistochemistry-positive).…”

“…3,9,13,[19][20][21] The importance of TP53 mutations has been validated in myelodysplastic syndrome in the non-therapy-related setting, particularly in myelodysplastic syndrome with del(5q). 21,22 One recent study showed that TP53 mutations in therapyrelated myeloid neoplasms predicted an inferior clinical outcome compared with wild-type TP53, but the authors examined a relatively small number of cases. 15 Complete assessment of TP53 mutation requires sequencing of all exons, because mutations often occur outside of the most commonly recognized hot spots, and requires a highly sensitive assay, because the mutation may only be present in a subset of the hematopoietic cells.…”