The Prognostic Impact of Epidermal Growth Factor Receptor (EGFR) in Patients with Acute Myeloid Leukaemia

Abstract

Expression of Epidermal Growth Factor Receptor (EGFR), an important proto-oncogene, regulates cell differentiation, proliferation, cell migration and survival in most of the cancer types. EGFR expression has been reported in Acute Myeloid Leukaemia (AML), however, many other reports nullified EGFR expression in AML. These contradictory data prompted us to reevaluate the expression of EGFR in AML and carry out a comparative survival analysis between EGFR expressing and non-expressing AML patients (Children and Acute Promyelocytic Leukaemia patients excluded). Bone marrow and/or peripheral blood samples were collected from 60 adult patients with AML with written informed consent. PCR, Real-Time Taqman gene expression assays were used for the detection of genetic alterations. Statistical analysis was conducted using SPSS software (IBM SPSS 20). In our study, EGFR expression was detected in 21 out of 60, in 35% (95% C.I. 23.45–48.48) AML patients. Overall survival was significantly shorter in patients with EGFR (p = < 0.01), with an average survival of 18.57 months (95% C.I. 12.42–24.73 months) compared with 31.27 months (95% C.I. 28.19–34.33 months) in patients without EGFR. EGFR expression was significantly higher in female patients compared to male (p = 0.037).This study confirms the presence of EGFR in AML and indicates that EGFR expression confers poor prognosis in AML. However, the underlying cause of this adverse prognostic effect has not been identified. Further clinical studies are warranted to determine the exact mechanism through which EGFR activity might contribute to AML progression and identify the potential therapeutic target for the reversal of resistance to conventional chemotherapeutics.

Introduction

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous clonal disorder that results in disruption of self-renewal, proliferation, and differentiation. Molecular and cytogenetic studies have reported a number of genetic alterations in AML such as Runt-related transcription factor 1/RUNX1 Partner Transcriptional Co-Repressor 1(RUNX1-RUNX1T1), Core-Binding Factor Beta/Myosin Heavy Chain 11 (CBFβ-MYH11) and Fms-like Tyrosine Kinase 3 (FLT3), TP53, IDH mutations having varied prognostic impacts [1]. Expression of Epidermal Growth Factor Receptor (EGFR), an important proto-oncogene that regulates cell differentiation, proliferation, cell migration, and survival has been reported in AML. EGFR is a cell surface receptor belonging to the ERBB family of proteins. Binding of ligand (EGF) activates EGFR through dimerization leading to activation of other downstream pathways such as Ras/Raf/Mitogen-activated protein kinases (Ras/Raf/MAPK), phosphatidylinositol 3-kinase/Akt (PI3K/Akt) through signal transduction [2]. Mutations in EGFR lead to constitutive receptor activation. Overexpressed EGFR results in aberrant signaling that induces uncontrolled cell growth and oncogenesis [3]. The expression of EGFR is not uniform across all the cell types. It has been shown that EGFR is expressed in epithelial, mesenchymal and neuroectodermal cells but not in hematopoietic cells [4]. Recent studies have reported EGFR gene expression in AML and emphasized on the fact that EGFR inhibitors in combination with other potential drugs may prove to be useful against the haematological malignancy [5], while others have reported no EGFR expression in AML cells [6].

The expression of EGFR in AML is poorly defined and the role of EGFR in AML remains unclear which prompted us to reevaluate the expression of EGFR in patients with AML and carry out a comparative survival analysis between EGFR expressing and non-expressing AML patients (Children and Acute Promyelocytic Leukaemia patients excluded).

Materials and Methods

Sample Collection

Bone marrow aspirate or peripheral blood samples were collected in 2 ml K2EDTA tubes (Becton–Dickinson, Franklin Lakes, NJ, USA), from 60 de novo adult AML patients with written informed consent. These tubes were mixed well and transported to the laboratory on ice as soon as possible and processed within 24 h of collection. Ethics committee clearance was obtained from the institutional ethics committee to conduct the study.

Cytomorphology and Karyotyping

As per standard techniques bone marrow and peripheral blood, smears were stained following French-American-British (FAB) and World Health Organization (WHO) criteria [7]. Karyotyping was done at diagnosis according to the International System for Human Cytogenetic Nomenclature [8].

Molecular Assays

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) extraction and cDNA preparation, FLT3 (FLT3-ITD and FLT3-D835), NPM1 mutation detection were performed as discussed earlier [9]. FLT3-ITD was detected by PCR amplification (Veriti, Thermo Fisher Scientific) whereas FLT3-D835 mutation was detected by PCR amplification and amplified products were then digested with EcoRV restriction endonuclease (HiMedia Laboratories Pvt. Ltd, Mumbai, India). NPM1 mutations were detected using PCR amplification followed by Sanger sequencing (sequence outsourced). The primers for screening TP53 mutations in exons 5–9 were used as described before [10]. TP53 mutations were detected using PCR amplification followed by Sanger sequencing. IDH mutation detection was performed as described earlier [11]. IDH mutations were detected using PCR amplification followed by Sanger sequencing. Taq-man assays (Taqman Universal Master Mix II with UNG from Thermo Fisher Scientific Waltham, MA USA) were used in Real-Time PCR to detect RUNX1-RUNX1T1 (ID: Hs03024752_ft), CBFβ-MYH11 (ID: Hs03460064_ft) translocations and EGFR (ID: Hs01076078_m1) expression in a StepOnePlus (Thermo Fisher Scientific Waltham, MA USA) Real-Time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as endogenous control (ID: Hs99999905_m1) with all assays.

Statistical Analysis

The main focus of our statistical analysis was to determine the significance of the overall survival between the EGFR expressing and non-expressing AML patients. Kaplan–Meier survival curve was constructed to explain the survival status between EGFR positive and negative patients. For statistically significant, p value < 0.05 was considered. SPSS software (IBM SPSS 20) was used for the analysis.

Treatment Protocol

At present, the standard treatment paradigm for AML is induction chemotherapy with an anthracycline/cytarabine combination followed by either remission consolidation therapy or allogeneic stem cell transplantation. Patients received remission induction therapy “7 + 3 regimen” of cytarabine (100 mg/m²) for 7 days plus daunorubicin (60 mg/m²) for 3 days. As a consolidation 3 cycles of high dose cytarabine 3 g/m² every 12 h on days 1, 3 and 5 were given.

Results

In our study group, we detected EGFR expression in 21/60, 35% (95% C.I. 23.45–48.48) AML patients. Comparison of 21 EGFR expressing and 39 non expressing AML did not reveal any statistically significant difference in clinical parameters such as age, white blood cell (WBC) count, platelet, haemoglobin count, blast count, and prevalence of common genetic alterations at diagnosis (Table 1). CBFβ-MYH11, FLT3-D835 and IDH mutations were not detected in the study group.

Table 1.

Clinical characteristic of EGFR positive and EGFR negative patients

Comparison between EGFR positive (n = 21) and EGFR negative (n = 39)
Demographic, laboratory and clinical characteristics	+ve	-ve	P value
Gender	n (%)	n (%)	< 0.05
Male	7 (22.6%)	24 (77.4%)
Female	14 (48.3%)	15 (51.7%)
Sex ratio (M:F)	0.5	1.6

	EGFR (+ve) Mean ± SD [median(range)]	EGFR (−ve) Mean ± SD [median(range)]	P value
Age (years)	36 + 8.64 [34 (28–48)]	44.37 + 13.84 [42(25–84)]	> 0.05
WBC count, × 109/L	116911.43 + 219703.64 [25,000 (10,640–957,000)]	47843.95 + 56765.63 [25,830(5600–224,500)]	> 0.05
Platelet count, × 109/L	58714.29 + 117558.56 [30,000 (5000–567,000)]	42962.05 + 47151.34 [26,000 (4000–215,000)	> 0.05
Haemoglobin level, × g/dL	6.86 + 1.91 [7.0 (4–10)]	7.10 + 2.42 [7.0 (2–13)]	> 0.05
Blast count (%)	44.14 ±20.29 [39 (20–80)]	47.05 + 20.44 [45 (20–85)]	> 0.0

	EGFR (+ve) n (%)	EGFR (-ve) n (%)	P value
FLT3-ITD	2 (9.5)	2 (5.1)	> 0.05
RUNX1-RUNX1T1	4 (19)	6(15.4)	> 0.05
NPM1	8 (38.1)	15 (38.5)	> 0.05
IDH	0 (0)	0 (0)	–
TP53 mutation	1 (4.8)	2 (5.1)	> 0.05
Dead patients	12 (57.1)	5 (12.8)	< 0.01

P values computed using Pearson Chi square, Unpaired and Mann–Whitney U tests

From the KM-curve (Fig. 1) it has been observed that EGFR positive patients survive on the average 18.57 months (95% C.I. 12.42–24.73 months) whereas EGFR negative patients survive on the average 31.27 months (95% C.I. 28.19–34.33 months). The result of the Log-rank test shows that the survival time between EGFR positive and negative patients is significantly different (p value: 0.000178). The proportion of EGFR positive among males was 22.6% whereas in the case of females every 48 females out of 100 females were EGFR positive. The proportion of EGFR positive female was significantly higher than the male counterpart (p value: 0.037).

Fig. 1.

Survival analysis between EGFR positive and negative patients in AML

Discussion

Despite the critical role of EGFR in maintaining the normal cellular function, regulation of EGFR signaling pathways is important since dysregulation of EGFR signaling pathways triggers the development of malignancy via effects on cell-cycle progression, inhibition of apoptosis, induction of angiogenesis, and promotion of tumor-cell migration [12]. EGFR plays a pivotal role in the pathogenesis and progression of various cancers, mainly in solid tumors [13]. In cancers such as breast cancer, bladder cancer, glioblastoma multiforme, non-small cell lung cancer the involvement of EGFR is already been studied [14]. Aberrant expression of the EGFR gene leads to poor prognosis in different cancer types [15]. On the contrary, the view on EGFR expression in AML is highly debatable and poorly defined which prompted us to carry out this study.

To the best of our knowledge, this is the first report from India relating to the clinical outcome of EGFR in patients with AML. Our study shows that the frequency of EGFR in AML is almost 35% and confers a poor prognosis. The presence of an EGFR mutation does not respond well to conventional chemotherapy. Targeted therapies to EGFR offers the promise of better treatment for different types of solid tumors. Despite EGFR inhibitors such as gefitinib (Iressa), erlotinib, 4, 5-dianilinophthalimide (DAPH1) were found to induce myeloid maturation and inhibit viability in AML cell lines, as well as in primary patient AML blasts, at low micromolar concentrations [5], there have been no clinical trials published evaluating the efficacy of EGFR inhibitors in patients with AML and hence a potential therapeutic target for the reversal of resistance to conventional chemotherapeutics needs to be identified.

Conclusion

This study confirms the presence of EGFR in AML and also indicates that EGFR expression confers poor prognosis in AML. There are not enough clinical data at present to make an evidence-based clinical decision for AML patients with EGFR and conventional chemotherapeutics is not sufficient enough to accurately represent all the resistance mechanisms. Further clinical studies are warranted to determine the exact mechanism through which EGFR activity might contribute to AML progression and identify the potential molecules to target to overcome drug resistance.

Author’s Contribution

All authors listed have contributed to the work and agreed to submit the manuscript for publication

Funding

This study was financially supported by a grant from Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India (Grant Number BT/348/NE/TBP/2012)

Compliance with Ethical Standards

Conflict of interest

Sukanta Nath, Jina Bhattacharya, Partha Pratim Sarma, Renu Saxena, Sudha Sazawal, Manash Pratim Barman and Kandarpa Saikia declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Study methodology was approved by the institutional ethics committee of Gauhati University, Guwahati, Assam, India (GUEC-12/2015).

Informed Consent

Informed consent was obtained from all individual participants included in the study