Characterization of <i>TP53</i> mutations in clonal cytopenia of undetermined significance
Authors: Wei Wang, Mark J. Routbort, Chi Young Ok, Keyur P. Patel, Yi Sun, Rashmi Kanagal‐Shamanna, L. Jeffrey Medeiros, Sa A. Wang
Journal: American Journal of Hematology (2017)

The diagnosis of myelodysplastic syndrome (MDS) requires persistent cytopenia with at least one of the following criteria: dysplasia in at least 10% of cells in any hematopoietic lineage, increased myeloblasts (5–19%) in bone marrow (or 2–19% myeloblast in peripheral blood), or MDS defining cytogenetic abnormalities. Some patients have cytopenia and/or gene mutations, but do not meet other criteria of MDS.1 These pre-MDS conditions include idiopathic cytopenia of undetermined significance (ICUS), clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). The mutations frequently identified in these pre-MDS conditions, including DNMT3A, TET2, and ASXL1, are also the common mutations detected in MDS.2 ICUS, CHIP, and CCUS all carry an increased risk for progression to MDS. The rate of progression to MDS varies, likely depending on the specific genes that are mutated and their mutation burden. The role of each individual mutation in disease progression is not well characterized. TP53 is a tumor suppressor gene that has been studied extensively in MDS and AML, in which the mutations are associated with a complex karyotype and a poor prognosis. Its mutations also occur in CHIP and CCUS.2, 3 The characteristics of TP53 mutations and their role in disease progression in these pre-MDS conditions are unknown. In this study, we aim to characterize the clinicopathological features of CCUS cases associated with TP53 mutations. Cases were selected for this study based on two criteria: 1, patients presented for an MDS workup due to persistent cytopenia; 2, TP53 gene mutations were detected using next generation sequencing-based methods. In total, 184 patients were identified to have concurrent cytopenia and TP53 mutations, and 176 (96%) had a definitive diagnosis of MDS. The remaining 8 (4%) patients did not meet the diagnostic criteria for MDS as they did not have morphological dysplasia, increased blasts, or MDS defining cytogenetic changes. In all 8 patients, bone marrow aspirate specimens assessed by flow cytometry immunophenotyping showed CD34+ myeloid precursors with a normal immunophenotype and conventional cytogenetic analysis showed a normal karyotype. Based on persistent cytopenia and evidence of a clonal process (TP53 mutations), they were diagnosed with CCUS. This CCUS group included 4 (50%) men and 4 (50%) women with a median age of 69 years (range, 44–83) at the time that TP53 mutations were detected. All patients had a history of malignancies and corresponding therapies, including chemotherapy, radiation, or immunotherapy (Supporting Information Table 1). All patients underwent bone marrow examination due to prolonged cytopenia and a high clinical suspicion of MDS. Similar to MDS cases, TP53 mutations in CCUS cases also occurred in DNA binding domain (exons 5–8) (Supporting Information Figure 1). In total, nine TP53 mutations were identified in 8 patients; 2 in exon 5 (22%), 3 in exon 6 (33%), 1 (11%) in exon 7 and 3 (33%) in exon 8. One TP53 mutation was present in 7 of 8 (88%) CCUS cases, comparable to the rate (128/176, 73%) detected in MDS cases (P = .68, Fisher's exact test). As shown in Figure 1A, the median variant allele frequency (VAF) of TP53 mutations was 3.7% (range 1.7%–16.8%) in CCUS cases, significantly lower than the rate detected in MDS cases (median, 36.5%; range, 1%–93.9%) (P < .0001). (A) VAF of TP53 mutations in CCUS and MDS cases. (B) Case #7: VAF of TP53 mutations, karyotypes, blast counts, morphology, and flow cytometric immunophenotype in different stages of disease progression. Left, CCUS; middle, MDS, and right: AML [Color figure can be viewed at wileyonlinelibrary.com] Seven patients in this study group had follow-up for peripheral blood counts. The median follow-up was 7 months (range, 0.3–18.1). Two patients also had a repeat bone marrow examination during the follow-up interval, in case #7 due to worsening cytopenia and in case #5 due to the potential interference by growth factor administration in the initial biopsy. The remaining cases did not have a second bone marrow examination because of stable or improved peripheral blood counts. Patient #7 (Supporting Information Table 1), who showed worsening cytopenia during follow-up, developed therapy-related MDS 8 months after the initial diagnosis of CCUS. The patient did not respond to decitabine treatment and 1.6 months later he progressed to AML. The VAF of TP53 mutation increased from 6.9% in CCUS, to 14.4% in MDS, and to 41.1% in AML. The karyotype was normal in CCUS and became complex in AML. The karyotype at the time of MDS diagnosis was unknown due to insufficient number of metaphases harvested. The morphologic features are illustrated in Figure 1B. In comparison with the bone marrow at time of CCUS, MDS marrow showed increased blasts (11%) with significant erythroid dysplasia, which was absent in CCUS specimen. Flow cytometric analysis revealed multiple immunophenotypic aberrancies in CD34+ myeloblasts in the MDS and AML specimens that were not present in the CCUS specimen (Figure 1B). The aberrancies included decreased/absent of hematogones (normal immature B cell precursors), increased CD4 expression, decreased HLA-DR expression, and expression of CD15. The presence of a low VAF in CCUS, higher VAF in MDS and AML, and the association between a rising VAF and disease progression suggest that the expansion of TP53 mutated clone is responsible for the development and progression of therapy-related myeloid neoplasms. In the remaining six patients, three (cases #1, 3, 6) showed persistent cytopenia, and three (cases #2, 5, 8) had improved blood counts. Their complete blood counts at the initial diagnosis and latest follow-up are listed in Supporting Information Table 1. It is of interest that all CCUS patients in our cohort had a history of malignancy that had been treated with cytotoxic therapy (Supporting Information Table 1), indicating a potential association between prior therapy and the emergence of TP53 mutations. Recent studies have shown that cytotoxic therapy can enrich hematopoietic stem cells that possess pre-existing genetic mutations. Cells carrying mutations are resistant to chemotherapy and expand during and after treatment.4 Other conditions, such as autoimmune diseases, inflammation/infection, and immunomodulating drugs have also been proposed as providing a favorable environment for mutant cells to grow and expand.5 In support of this theory, in one study 47% of patients with aplastic anemia were found to have clonal hematopoiesis.6 In the current study, it seems likely that TP53 mutations were already present prior to treatment at a very low VAF, which expanded after cytotoxic therapy as they had survival and growth advantages. In summary, similar to MDS, TP53 mutations in CCUS show similar sites and numbers. In contrast to MDS, the VAF of TP53 mutation in CCUS cases is low, often 5% or less, and a rise in VAF is associated with disease progression. A history of malignancy and cytotoxic therapy in these patients indicates an association between prior therapy and the emergence of TP53 mutations, resulting in an expansion of CHIP clones. Patients with CCUS associated with TP53 mutations can progress to MDS/AML and need a close follow-up. Additional Supporting Information may be found in the online version of this article. Supporting Information Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.