- © 2013 by American Society of Clinical Oncology
Activating Germline R776H Mutation in the Epidermal Growth Factor Receptor Associated With Lung Cancer With Squamous Differentiation
- Corresponding author: Carel J.M. van Noesel, MD, PhD, Professor of Molecular Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; e-mail: c.j.vannoesel{at}amc.uva.nl.
Introduction
Somatic mutations that affect the adenosine 5′-triphosphate–binding pocket of the kinase domain of the epidermal growth factor receptor (EGFR) are found in approximately 10% and 25% of non–small-cell lung cancers (NSCLCs) in the Western world and East Asia, respectively,1 most often in adenocarcinomas and bronchioloalveolar carcinomas. NSCLCs with EGFR gene mutations and/or amplifications are hypersensitive to EGFR tyrosine kinase inhibitors and, concordantly, may display a favorable clinical response and improved survival after treatment with gefitinib and erlotinib.2
Case Reports
We analyzed a mother and daughter of a white family. Neither of the patients smoked, and both patients were diagnosed with lung cancer. In the mother, a tumor was detected in the right hilar region in July 2004 at age 57 years. A bronchoscopically obtained biopsy and subsequent cytologic specimens derived of a lymph-node metastasis and pleuritis carcinomatosa revealed an NSCLC with apparent squamous differentiation (Fig 1A). The patient was treated with chemotherapy and local radiotherapy. In October 2005, brain metastases became evident, and the patient died 2 months later.
The daughter was diagnosed with right-sided lung cancer at age 36 years in March 2009. The histologically assessed diagnosis was squamous-cell carcinoma (Fig 1B), which was confirmed immunohistochemically as being thyroid transcription factor-1 negative (Fig 1C) and p63 positive (Fig 1D). A cytologic puncture of a locoregional lymph node demonstrated metastatic disease. The patient was treated with chemotherapy and local radiotherapy followed by a lobectomy with mediastinal lymfadenectomy. Sequence analysis of EGFR on tumor-derived DNA revealed the copresence of two previously reported missense mutations in exon 18 [c.2155G>A, p.(G719S)] and in exon 20 [c.2327G>A, p.(R776H); Fig 2A; T]. After the lobectomy, adjuvant treatment with the EGFR-inhibitor erlotinib was started. The patient remained free of detectable disease after 1 year of anti-EGFR therapy.
Sequence analysis further revealed that the codon 776 mutation was also detectable in DNA derived from normal lung tissue (Fig 2A; N), normal lymph node, skin, and blood and, thus, probably in the germline and was inherited from one of her parents, most likely her mother. Subsequent analyses on cytologic specimen derived from the lymph node of her mother revealed that the R776H mutation of EGFR was indeed present in the DNA of both tumor cells and normal lymphoid cells (Fig 2B; N). Interestingly, in the carcinoma of the mother, a second EGFR mutation was found in codon 719 (c.2156G>C), which resulted in a glycine to alanine substitution [p.(G719A); Fig 2B; T]. This missense mutation was not present in her normal DNA. With respect to other family members, only the brother of the mother was tested and was found not to carry the codon 776 mutation.
Chromogenic in situ hybridization on tissue sections (Figs 3A, 3B) by using labeled DNA probes specific for the EGFR gene locus (black dots) and for the centromeric region of chromosome 7 (red dots) revealed, for both probes, an equally increased number of signals in the tumor cells of the daughter. On average, from three to four separate signals, and no clusters, were found in every tumor cell (Fig 3B) compared with two signals in non-neoplastic stromal cells that surrounded the tumor fields (Fig 3A). This finding was consistent with a gain in copy number of (parts of) chromosome 7 (polysomy) and not with EGFR gene amplification. Unfortunately, we could not analyze the tumor of the mother by using chromogenic in situ hybridization as a result of a lack of remaining paraffin-embedded tissue.
The combined EGFR gene mutations in tumor cells may occur within the same or separate alleles, which has implications for the receptor complexes at the cell surface. To address this issue, we amplified, by reverse transcriptase polymerase chain reaction, cloned and sequenced individual EGFR gene transcripts that spanned the regions from exons 18 to 21. Of 27 sequenced clones derived from tumor of the daughter, 13 clones were unmutated, whereas 14 sequences contained both codon 719 and 776 mutations. From RNA of the tumor of the mother, 26 clones were sequenced, of which 11 clones were unmutated, and 15 clones harbored both mutations. Thus, it was concluded that, in the tumors of the mother and daughter, germline and somatic mutations were on the same EGFR alleles.
Wild-type (WT) and mutant EGFRs were analyzed in vitro for kinase activity, the ability to confer growth factor independence, and their sensitivity to different inhibitors. Human embryonic kidney 293 cells, which lack endogenous EGFR (UT), were transfered with WT or mutant EGFR. Western-blot analysis of two separate gels showed EGFR autophosphorylation (P-EGFR) induced by EGF (Fig 4A; WT positive; ERK 1/2, extracellular signal-related kinases 1/2; PY, thyrosin phosphorylation; STAT5, signal transducer and activator of transcription 5). In the G719A and R776H single mutants, constitutive, ligand-independent activation was observed (Fig 4A; G719A negative and R776H negative), and levels of EGF-induced phosphorylation (Fig 4A; G719A positive and R776 positive) were significantly higher than those of WT EGFR. The analysis also revealed enhanced constitutive and ligand-induced PY, specifically of the downstream prosurvival and proliferation mediators ERK and STAT5, in cells that carry the mutant EGFRs compared with those with WT EGFRs. For the G719S single mutant, a similar enhancing effect on EGFR activity has been described previously by our group.3 The enhancing effect of the R776H/G719A and R776H/G719S double mutants on autonomous and EGF-induced pathway activation, compared with the single mutants, was, at best, additive but not synergistic.
We next established EGFR-mutant–expressing Ba/F3 cell lines, which in the absence of interleukin-3 completely depend on the overexpressed EGFR for their survival. Ba/F3 cells transduced with the (double)-mutant EGFR proliferated in the absence of interleukin-3. This was also true for the R776H mutant (data not shown), which confirmed the oncogenic potential of this variant found in the germline of the patients. The R776H/G719A and R776H/G719S double mutants retained sensitivity to the clinically approved EGFR inhibitors gefitinib and erlotinib (Fig 4B). The R776H/G719A-expressing cells displayed somewhat higher inhibitory concentration 50% (IC50) values against gefitinib (346 nmol/L) and erlotinib (140 nmol/L) compared with R776H/G719S (156 and 28 nmol/L against gefitinib and erlotinib, respectively). The lower IC50 value of R776H/G719S may have been attributed to the effects of G719S because this mutation was previously shown to confer inhibitor-sensitizing effects.3–5
Discussion
The finding that, in both tumors, the secondary somatic mutation was in codon 719 was striking because codon 719 mutations are uncommon. In the Catalogue of Somatic Mutations in Cancer of the Wellcome Trust Sanger Institute6 somatic codon, 719 mutations are found in less than 0.5% of all EGFR-mutated lung carcinomas included (> 27,000 cases). Mutations in codon 776 were reported in a total of 12 carcinomas and altered the arginine in a histidine, cysteine, or glycine. The R776H (c.2327G>A) mutation was reported in only eight carcinomas.7–12 Interestingly, in all eight patients, the R776H was found together with a second EGFR mutation, four of which were the G719A mutation,9 and three were the common L858R or L858Q mutations.11,12 Moreover, because the somatic origin of the R776H mutation was not assessed in any of these carcinomas, it could not be excluded that these patients might also carry the R776H EGFR variant in the germline. If so, this would suggest that this germline variant has a frequency of approximately 0.4 ‰ among patients with EGFR-mutated NSCLCs.
Previously, two germline-transmitted EGFR variants have been reported to associate with lung cancer, ie, the T790M mutation,13,14 which is also known to confer tyrosine-kinase inhibitor resistance, and the V843I mutation, which is associated with multicentric adenomatous hyperplasia and adenocarcinomas.15 In a proportion of carcinomas in patients carrying these germline mutations, somatic mutations were present in cis with the germline variant. In more than 50% of cases, this involved the common L858R mutation (occurring in 45% of sporadic EGFR-mutated lung cancers). Interestingly, an NSCLC has recently been reported harboring a germline-derived R776G mutation in combination with an L858R mutation.16 The combination of R776G and L858R mutations had also been described in another adenocarcinoma.11 The somatic status of the mutations, however, was not assessed in the latter study. Finally, in one patient with the T790M germline mutation, the secondary somatic mutation was the G719A.13 This finding suggests that, in patients carrying an EGFR germline mutation, the secondary transforming events in the same EGFR gene allele were the outcomes of stringent selection for EGFRs with an appropriate level of spontaneous kinase activity.
In addition to the fact that the tumors of the mother and daughter shared the R776H and codon 719 EGFR mutations, it was noticeable that both appeared as squamous-cell carcinomas, one of which was immunohistochemically proven (Figs 1C and 1D). Within large cohorts of EGFR-mutated lung carcinomas, the vast majority of them are classified as bronchioloalveolar carcinomas or adenocarcinomas, although squamous- or mixed adenosquamous-cell carcinomas represent approximately 1% of them. In the database mentioned, one of the four R776H/G719A-mutated tumors was also classified as mixed adenosquamous-cell carcinoma.9 In addition, more than 10% of carcinomas with somatic codon 719 mutations were designated as (mixed adeno) squamous carcinomas, in particular carcinomas with a G719S mutation (15%). Thus, the squamous nature of the tumors in our two patients may have been related, to an uncertain extent, to the combination of mutations they carried. As yet, is hard to envisage how mutations that merely activate the kinase domain of EGFR might have differential effects on tumor-cell differentiation.
AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
The author(s) indicated no potential conflicts of interest.
ACKNOWLEDGMENT
We thank M.J. de Wit, A.R. Musler, M.M. Polak, and G.K.J. Hooijer for expert technical support and Dr J.E.J. Guikema for discussion and critically reading the manuscript. Both J.v.N. and W.H.v.d.V. contributed equally to this work.
