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TP53 mutations linked to increased immune infiltration and IFN-γ signaling in patients with AML

Dec 4, 2020
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Patients with acute myeloid leukemia (AML) are considered to be in the high cytogenetic risk category if they harbour TP53 mutations of 17q deletions that lead to complete loss of TP53.1 These patients have very poor outcomes and are not generally good candidates for hematopoietic stem cell transplantation (HSCT) due to advanced age or comorbidities.1 This automatically further reduces their potential for survival, as HSCT is a curative option for many patients with AML.1 Mutations in the tumor-suppressing gene TP53 have recently been shown to correlate with increased white blood cell infiltration in various cancers.1 Jayakumar Vadakekolathu and colleagues1 investigated the role of TP53 mutations in forming the immune landscape and microenvironment in AML. The results of this computational study were published in Blood Advances and are summarized below.

Study design

  • Immune gene expression profiling was performed in bone marrow (BM) samples from three distinct patient cohorts and from The Cancer Genome Atlas (TCGA) genomic database.
  • Patient cohorts:
    • Studien-Allianz Leukämie (SAL) cohort (N = 40): BM samples from patients with newly diagnosed (ND) TP53-mutated AML treated with curative intent.
    • Bologna cohort (N = 24): BM samples from patients with ND AML treated with curative intent.
    • CP-MGD006-01 clinical trial cohort (N = 45): BM samples from patients with primary induction failure or early/late relapse AML treated with flotetuzumab (500 ng/kg daily) in the CP-MGD006-01 trial (NCT02152956).
  • Patients with prior HSCT were not included in the study.
  • Patient baseline characteristics are shown in Table 1. In the SAL cohort, all patients harboured TP53 mutations, while in the Bologna cohort the majority were wild type with only two TP53-mutated patients. In the CP-MGD006-01 clinical trial cohort, 15 patients harboured either TP53 mutations or 17q deletions.
  • For the in vitro experiments, the following AML cell lines were used:
    • Kasumi-1: these cells harbour the R248Q missense TP53 mutation.
    • KG-1: these cells harbour a defective donor splice site in intron 6 of TP53 leading to loss of function of the TP53 protein.

Table 1. Patient baseline characteristics1

 

Patient samples

Genomic database

 

Baseline characteristic

SAL

Bologna

CP-MGD006-01 trial

TCGA

 

AML, acute myeloid leukemia; BM, bone marrow; ELN, European LeukemiaNet; HMAs, hypomethylating agents; MAV, mitoxantrone, cytarabine, etoposide; NA, not available; OS, overall survival; SAL, Studien-Allianz Leukämie; TCGA, The Cancer Genome Atlas; WBC, white blood cell; WT, wild-type.
*Patients with either TP53 mutations or 17q abnormalities. Only 13 out of 15 BM samples were available for gene profiling, but all 15 patients were included in analysis.

 

No. of patients 

40

24

45

147

 

Males/females, n 

26/14

17/7

24/21

81/66

 

Age, n 

 

 

  0−14 years 

0

0

0

 

  15−39 years

2

3

27

 

  40−59 years

13

12

45

 

  > 60 years

25

9

75

 

Median age, years (range)

61 (27−81)

 

Median WBC count at presentation, × 103/μL (range)

10.55
(0.8–218.5)

45.0
(1.5–153.0)

20.0
(1.0–297.0)

 

Median BM blasts, % (range)

63.7
(30.0–90.0)

16.5
(0.4–57.0)

72.0
(11.0–99.0)

 

ELN cytogenetic risk group, n (%) 

 

 

  Favorable 

0 (0)

6 (26.1)

3 (6.7)

17 (12.0)

 

  Intermediate 

0 (0)

7 (30.4)

8 (17.8)

96 (65.0)

 

  Adverse 

40 (100)

5 (21.7)

34 (75.6)

32 (22.0)

 

  NA 

0 (0)

5 (21.7)

2 (1.0)

 

TP53 status, n

 

 

 

  Mutated 

40

2

15*

14

 

  WT 

0

22

30

 

  Not tested/not available 

133

 

Induction chemotherapy 

 

 

  7 + 3 

5

2

113

 

  Fludarabine-based 

8

 

  Daunorubicin + cytarabine 

21

0

 

  MAV 

12

5

 

  HMAs 

3

14

 

  Lenalidomide 

1

9

 

  Other 

1

6

11

 

Median cohort-wide OS, months (range) 

5.06
(0.03–158.30)

16.50
(0.30–57.00)

15.50
(0.10–118.10)

 

Median number of prior lines (range)

2 (1−9)

 

Secondary AML, n (%)

15 (33.3)

 

Results

In silico analysis

  • Initial screening in TCGA genomic database retrieved 147 cases with both RNA-sequencing and clinical data from adult patients with ND non-promyelocytic AML. Cases with concomitant TP53 and DNMT3A or TET2 mutations were excluded from the analysis as such mutations are already known to be prognostic in AML.
  • The analysis between wild-type and TP53-mutated AML cases showed that TP53-mutated samples had:
    • Increased immune infiltration, as observed by increased interferon (IFN)-γ signaling, increased tumor inflammation signature (TIS), and increased expression of chemokine- and lymphoid-related mRNAs.
    • A higher number of mutations.
    • A higher proportion of genomic alterations.
    • A higher expression of negative immune checkpoint regulators (i.e. PDL1 and FoxP3) and genes associated with CD8+ T-cell, Treg and other immune cell infiltration in the BM microenvironment.

Primary BM sample genetic analysis

Primary BM samples extracted from the SAL and Bologna cohorts were compared in terms of their immune gene signature (TP53-mutated AML: n = 42; wild type AML: n = 22). Principal component analysis of 770 immunomodulatory genes and cancer pathways were explored to identify those associated with TP53 mutations that may also be prognostic of patient outcomes.

  • Based on the genetic signatures identified in the principal component analysis above, it was possible to distinguish wild-type from TP53-mutated samples.
  • In patient samples with high, intermediate, or low immune infiltration signatures, the frequency of TP53 mutations was 87%, 75%, and 8%, respectively.
  • When compared to wild-type, TP53-mutated samples had higher expression of genes involved in the nuclear factor-κB (NF-κB), Janus kinase-signal transducer and activator of transcription (JAK-STAT), and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways, but lower expression of DNA damage repair signaling genes, like Wnt and hedgehog.
  • In total, 34 differentially expressed immune genes were identified between TP53-mutated and wild-type samples. Genes overexpressed in TP53-mutated samples were classified as:
    • Neutrophil chemoattractants (proinflammatory CXCL1, CXCL2, CXCL8, and IL8).
    • IFN-inducible molecules (CCL2, IL33, IL6, OASL, and RIPK2).
  • The prognostic value of these 34 genes was assessed in TCGA AML cohort. Only the ones overexpressed (18 genes) were significantly correlated with TP53 mutational status (p = 0.041), increased immune infiltration, expression of immune checkpoints, and IFN signaling genes.

In vitro cell line experiments

To examine the effect of TP53 loss or mutation on IFN signaling, the KG-1 and Kasumi-1 cell lines were used.

  • The following genes were found to be overexpressed in each cell line:
    • KG-1 cells: overexpressed genes associated with IFN-γ signaling and inflammation (i.e., HGF, CIITA, PIM1, OSM, STAT1, and IRF1).
    • PDL1 and genes of the major histocompatibility complex class I were upregulated in KG-1 cells compared to Kasumi 1 cells.
    • Genes involved in the PI3K-Akt, NF-κB, JAK/STAT, and TP53 signaling pathways, and those linked to T helper 17 differentiation, were also enriched in KG-1 cells.
  • Overexpressed genes in KG-1 cells (loss of functional TP53) were predictive of shorter overall survival (OS; HR, 1.7; p = 0.048), while those upregulated in Kasumi-1 cells (TP53-mutated) were not prognostic.

Effect of flotetuzumab on TP53-mutated AML patient samples

The authors have previously shown that mRNA expression of INF-γ-associated genes, as well as TIS scores, have been associated with patient responses to flotetuzumab. To examine the potential link between TP53 mutations, BM immune signature profile, and complete response to flotetuzumab, the authors examined 45 patients from the CP-MGD006-01 clinical trial. Of those patients, 15 had either TP53 mutations or 17q abnormalities. Immune gene profiling was performed in 13 of these 15 patients with the following results:

  • Increased immune infiltration in the tumor microenvironment was observed in 77% of these patients.
  • Overall response rate was achieved by 60% of patients, with 47% of them having a complete response (CR).
  • The median OS of those achieving a CR following flotetuzumab was 10.3 months (range, 3.321.3).
  • Overall, patients with TP53-mutated (or TP53 loss of function) AML showed an insignificant trend of achieving a better overall response rate following flotetuzumab treatment compared to those with wild-type TP53 AML (60.0% vs 33.3%; p = 0.08).
  • Gene profiling analysis between flotetuzumab responders (n = 8) and non-responders (n = 10) showed that genes associated with TIS, chemokines, Treg, CD8, IFN-γ, and PD1 were significantly increased in responders at baseline. CD8D was the gene with the highest association to response to flotetuzumab (area under the curve = 0.879). Genes that were significantly downregulated in responders include CTNNB1 and ANGPT1.
  • Flow cytometry with wild-type and TP53-mutated (or loss of function) patient samples showed a higher number of CD4+ and CD8+ T cells in the BM of patients with TP53 genetic alterations, although this was not significant.

Conclusion

The results of this computational study indicate that patients with AML with TP53 mutations have higher levels of immune BM infiltration (especially CD8+ T cells), increased INF-γ-associated signaling, and higher expression of immune checkpoints. A specific gene signature profile of TP53-mutated samples, with overexpressed genes involved in IFN-associated, IL-17-associated, TNF-associated, and other inflammatory pathways were able to predict AML survival outcomes in silico. In vitro, genes overexpressed following TP53 loss of function (IFN-γ signaling molecules) were able to predict poorer survival outcomes. Lastly, patients with TP53 abnormalities who responded (CR) to flotetuzumab therapy had a good OS (10.3 months), indicating the potential use of flotetuzumab immunotherapy in poor risk TP53-mutated AML.

  1. Vadakekolathu J, Lai C, Reeder S, et al. TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immunotherapy in AML. Blood Adv. 2020;4(20):5011-5024. DOI: 10.1182/bloodadvances.2020002512

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