Cancer-testis antigen expression and its epigenetic modulation in acute myeloid leukemia

Introduction: In this single-center study of 268 acute myeloid leukemia (AML) patients, we have tested if a subset of 4 routinely employed immunophenotypic stem cell-associated markers correlated with the presence of recurrently mutated genes and if the markers were predictive for mutational status. Methods: Immunophenotypic data from 268 diagnostic AML samples obtained in 2009–2018 were analyzed retrospectively for the antigens CD34, CD117, CD123, and CLEC12A. Correlation between immunophenotypes and mutations was analyzed by Fischer’s exact test. Clinical applicability of the markers for predicting mutational status was evaluated by receiver operating characteristics analyses, where an area under the curve (AUC) of at least 0.85 was accepted as clinically relevant. Results: For a number of genes, the antigen expression differed significantly between mutated and wild-type gene expression. Despite low AUCs, CD123 and CLEC12A correlated with FLT3+NPM1− and FLT3+NPM1+. Three subsets met the AUC requirements (CD34+, CD34+CD117+, and CD34−CD117+) for predicting FLT3−NPM1+ or FLT3+NPM1+. Conclusion: The value of immunophenotypes as surrogate markers for mutational status in AML seems limited when employing CD123 and CLEC12A in combination with CD34 and CD117. Defining relevant cutoffs for given markers is challenging and hampered by variation between laboratories and patient groups.

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Lymphoid-Associated Antigen Expression by Acute Myeloid Leukemia

American Journal of Clinical Pathology ◽

10.1093/ajcp/106.2.185 ◽

1996 ◽

Vol 106 (2) ◽

pp. 185-191 ◽

Cited By ~ 12

Author(s):

Teresa M. Launder ◽

Robert A. Bray ◽

Linda Stempora ◽

Margie L. Chenggis ◽

Diane C. Farhi

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Antigen Expression ◽

Acute Myeloid

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Gene Mutations, Their Interactions and Associations with Immunophenotypes of Leukemia Cells in Patients with Primary Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.4138.4138 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4138-4138

Author(s):

Chien-Yuan Chen ◽

Hsin-An Hou ◽

Woei Tsay ◽

Jih-Luh Tang ◽

Liang-Inn Lin ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Gene Mutations ◽

Antigen Expression ◽

Class Ii ◽

Class I ◽

Leukemic Cells ◽

Leukemia Cells ◽

I Gene ◽

Acute Myeloid

Abstract The development of acute myeloid leukemia (AML) is a multistep process. Gilliland and colleagues proposed a two hit theory of leukemogenesis that requires collaboration of at least two classes of gene mutations. The Class I gene mutations activate the signal transduction pathway and confer proliferation and survival advantage to hematopoietic cells. The Class II gene mutations affect transcriptional activators or coactivators and serve to impair cell differentiation. In this study, comprehensive analyses of a panel of gene mutations, their interactions and associations with antigen expression of leukemia cells were performed in 324 patients with primary AML, including 275 adults and 49 children(≤18years). The gene mutations included FLT3/ ITD (78 cases, 24.1%), FLT/ TKD (24 cases, 7.4%), NPM(63 cases, 19.4%), CEBPA(45 cases, 13.9%), NRAS (39 cases, 12%), AML1 (31 cases, 9.6%), PTPN11 (14 cases, 4.3%), MLL/PTD(13 cases, 4%), KIT(10 cases, 3.1%), KRAS (8 cases, 2.5%), and JAK2 (3 cases, 0.9%). In addition, 33 patients had t(8;21), 24 had t(15;17), 9 had inv(16) and 13 had 11q23 translocations. Totally, the Class I gene mutations were detected in 155 patients (47.8%), and Class II gene mutations, in 228 patients (70.4%). Most Class II mutation was associated with a distinct immunophenotype of leukemic cells, such as CEBPA mutation: HLADR(+)CD7(+)CD15(+)CD19(−)CD34(+) (p<0.05), NPM mutation: HLADR(−)CD19(−)CD34(−)CD33(+)(p<0.05), AML1 mutation: HLADR(+)(p<0.05), MLL/PTD: CD7(−)(p<0.05), AML1/ETO: HLADR(+)CD7(−)CD19(+)CD33(−)CD34(+)CD56(+)(p<0.05), PML/RARA: HLADR(−)CD2(+)CD7(−)CD11b(−)CD34(−)(p<0.05), CBFB/MYH11: CD11b(+)CD14(+), and translocation 11q23: CD19(+)CD33(−)CD34(−) (p<0.05). The interactions between Class I and Class II mutations are shown in table 1. Among Class I mutations, FLT3/ ITD could interact with each subtype of Class II gene mutations, but were particularly associated with NPM mutations (p<0.001) and MLL/PTD (p=0.001). FLT3/ TKD was closely related to NPM mutations (p=0.03). Most KIT mutation were detected in the core binding factor leukemia (p<0.001). PTPN11 mutations were more frequently detected in patients with NPM mutations than in others (p=0.035). Few patients with complex cytogenetics revealed mutations of the gene panel studied (Table 1), suggesting that leukemogenesis in these patients was through mechanism other than the known Class I and Class II mutations. In this study, the cooperative gene alterations of the NUP98/HOXA9 fusion gene were demonstrated (Table1) which, to the best of our knowledge, have not been reported before. In conclusion, the development of AML requires multistep genetic changes. Most Class II mutation is closely associated with a distinct pattern of antigen expression of leukemic cells. Exploring the interactions of gene mutations may help us more understand the pathogenesis of leukemia and benefit further therapeutic strategy. Table I. Interaction of Class I and Class II gene mutations

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Induction of Cancer Testis Antigen Expression in Circulating Acute Myeloid Leukemia Blasts Following Hypomethylating Agent Monotherapy

Blood ◽

10.1182/blood.v126.23.2537.2537 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2537-2537

Author(s):

Pragya Srivastava ◽

Benjamin E. Paluch ◽

Junko Matsuzaki ◽

Smitha R. James ◽

Golda Collmat-Lai ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Promoter Methylation ◽

Myeloid Leukemia ◽

Off Label ◽

Tnf Α ◽

Ifn Γ ◽

Acute Myeloid ◽

Cancer Testis

Abstract Background: The development of immunotherapeutic strategies for the treatment of leukemia has shown considerable promise but targeting suitable myeloid leukemia antigens remains a significant challenge. Cancer testis antigens (CTA) have been identified as promising targets for immunotherapy in solid tumors, but dense promoter methylation silences their expression in acute myeloid leukemia, limiting their potential as targets. Patients with acute myeloid leukemia are frequently treated with hypomethylating agents (HMAs) and previous studies have established that exposure of leukemia cell lines to HMAs induces expression of CTAs. In this study, we tested the hypothesis that patients receiving HMAs exhibit increased expression of CTAs. Methods: AML patients receiving decitabine were enrolled under an IRB-approved protocol (Roswell Park) or with approval of the Ethics Committee (University of Freiburg). Peripheral blood samples were serially collected prior to decitabine treatment and two to four times per week during their first cycle of decitabine therapy (20 mg/m2 per day for 10 days). Results: We analyzed expression and demethylation of CTA genes in peripheral blood samples serially isolated from AML patients (n = 5) during and following their first cycle of decitabine therapy (20 mg/m2 per day for 10 days). These patients demonstrated induction of MAGEA1 (1/5 patients), XAGE1 (3/5), MAGEA3/A6 (3/5), and NY-ESO-1 (5/5). Western blot analysis demonstrated increased expression of NY-ESO-1 protein following decitabine treatment. The induction of NY-ESO1 mRNA was confirmed in an independent group of AML patients (5/7, treated at University of Freiburg) receiving decitabine. Since NY-ESO-1 is an established cancer immunotherapy target with clinically translatable vaccines in development, we further examined induced NY-ESO-1 expression in a larger cohort of AML patients. We performed sodium bisulfite pyrosequencing to quantify changes in NY-ESO-1 promoter methylation pre-decitabine to the post-decitabine nadir time point for each patient (n = 22). There was a statistically significant decrease in NY-ESO-1 promoter methylation (p < 0.001). Prior to decitabine treatment, 18% (4/22) of samples exhibited detectable levels of NY-ESO-1 mRNA as measured by quantitative PCR. Following decitabine therapy, 78% (17/22) of samples had detectable levels of NY-ESO-1 mRNA. Treatment with decitabine was associated with a significant increase in NY-ESO-1 expression when comparing pre-treatment expression to the maximum expression at any time interval post decitabine (p < 0.0001). We then tested whether levels of NY-ESO-1 induction were different in patients who demonstrated a clinical response compared with those who did not. Overall, 7/22 patients (32%) demonstrated a clinical response to decitabine. 6/7 patients who clinically responded to decitabine demonstrated a significant increase in NY-ESO-1 mRNA (p < 0.03). Crucially, NY-ESO-1 mRNA levels were also significantly increased in 11 out of the 15 patients that did not demonstrate a response to decitabine (p = 0.001). To test whether AML blasts expressing NY-ESO-1 could induce a T-cell response, we stimulated HLA compatible NY-ESO-1- specific CD8+ T cells with AML blasts isolated from HLA-A*0201+ AML patients before and after decitabine treatment. T-cell responses were determined by intracellular cytokine staining (IFN-γ, TNF-α and IL-2) and the expression of CD107a/b, a marker for T-cell degranulation. Co-culture of AML blasts harvested post-decitabine, resulted in increased levels of IFN-γ, TNF-α, IL-2, and CD107a/b in HLA-A*0201/NY-ESO-1157-165 tetramer+ CD8+ T-cells in three of the four patients studied, compared to T-cells co-cultured with AML blasts from the same four patients obtained prior to decitabine exposure. Conclusion: We observed enhanced expression of NY-ESO-1 in AML patients receiving decitabine and this induction was sufficient to produce a cytotoxic response in HLA-compatible antigen specific T-cells. A majority of patients who did not respond to decitabine still exhibited an increase NY-ESO-1 mRNA, suggesting that immunotherapies that target NY-ESO-1 have the potential to be effective even in patients who have not demonstrated a prior response to decitabine. These data support the combination of decitabine with immunotherapeutic approaches targeting NY-ESO-1 and other CTAs in myeloid cancer. Disclosures Griffiths: Alexion Pharmaceuticals: Honoraria; Astex: Research Funding; Celgene: Honoraria. Off Label Use: Decitabine is in routine clinical use in the United States for the management of unfit/elderly patients with AML. Patients received decitabine as standard of care off label in the hospital per Bloom et al PNAS 2010.

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