Novel EGFR V834L Germline Mutation Associated With Familial Lung Adenocarcinoma

Lung cancer is a worldwide, known disease with a high mortality rate and is often related to nicotine abuse. Family-related lung cancer has been reported occasionally, but little is known about the genetic backgrounds. With routine analysis for targetable mutations, unexpected potentially pathogenic mutations may be found. Here, we present a case study of a patient with a targetable somatic EGFR mutation and a novel EGFR germline variant, which also occurred in other family members with lung cancer.


INTRODUCTION
Lung cancer is a worldwide, known disease with a high mortality rate and is often related to nicotine abuse. Family-related lung cancer has been reported occasionally, but little is known about the genetic backgrounds. With routine analysis for targetable mutations, unexpected potentially pathogenic mutations may be found. Here, we present a case study of a patient with a targetable somatic EGFR mutation and a novel EGFR germline variant, which also occurred in other family members with lung cancer.

INDEX PATIENT
Our female index patient of Surinam origin, a former smoker with a 10 pack-year history, was diagnosed at the age of 57 years with nonsmall-cell lung cancer (NSCLC), stage IIIA cT2bN2M0 adenocarcinoma. Initial treatment included concurrent chemotherapy and radiotherapy, and palliative chemotherapy after recurrence (the treatment timeline is shown in Fig 1). After a second recurrence, a new biopsy of a lymph node metastasis was obtained for EGFR mutation analysis by next-generation sequencing. This analysis revealed a well-known oncogenic L858R mutation along with a V834L variant, which are both EGFR exon 21 substitutions and are present in the same allele (in cis). The V834L variant seemed to be present in the germline, because both normal adjacent lymph node tissue and lymphocyte-derived DNA from whole blood of the index patient showed this variant.
The patient was treated with the EGFR tyrosine kinase inhibitor (TKI) erlotinib. At the fourth progression of the disease, the patient was retreated with chemotherapy. After the fifth progression of the disease, a new biopsy revealed a first-generation TKI-resistance EGFR T790M mutation, in addition to L858R and V834L. Therefore, treatment was switched to the third-generation EGFR TKI osimertinib. Because of progression after 1 year of osimertinib treatment, a liquid biopsy was taken and cellfree DNA was isolated. Subsequent mutation analysis showed that the progression was probably because of the development of EGFR resistance mutation C797S in cis with T790M (data not shown). Performance of the patient was declining, and there were no remaining therapeutic options. Best supportive care was given, and the patient eventually died.

PATIENT'S FAMILY HISTORY WAS POSITIVE FOR LUNG CANCER
The patient's younger brother, who was 57 years old and a current smoker, was diagnosed in 2016 with stage IV, EGFR L858R mutation-positive NSCLC. He was treated with erlotinib, and his disease progressed after 3 months. A new biopsy was obtained, revealing no targetable secondary resistance mechanism, such as T790M mutation or MET amplification. Standard chemotherapy was started, but the patient died because of disease progression and decline in performance status.
A sister and daughter of our index patient, 46 and 42 years old, respectively, both nonsmokers, were diagnosed with stage IV NSCLC before the knowledge and treatment options of EGFR mutations. They were both treated with palliative chemotherapy but died as a result of disease progression. The patient's father died at a young age as the result of massive hemoptysis, without a diagnosis.
After written consent and permission were obtained from the index patient and family members, EGFR mutation and germline variation analyses were performed on tumor samples and healthy tissue, respectively, of the three affected relatives. Because our study started after case report the deaths of the index patient's brother, sister, and daughter, no blood was available; only archival formalin-fixed paraffin-embedded normal and tumor tissues were available.
Like our index patient, all three affected family members had a heterozygous EGFR V834L germline variant in normal tissue and the same somatic EGFR L858R mutation in cis with the V834L variant in their respective tumor tissues. No tissue samples from the patient's father were available.
Further germline research was performed in four healthy family members who did not have signs of lung cancer. One of the index patient's children, who was 36 years old, showed the same heterozygous EGFR V834L germline variant. All other investigated family members were wild type at this position in EGFR (Fig 2).

DISCUSSION
Little is known about hereditary lung cancer. This is probably in part because lung cancer is strongly associated with smoking and therefore a familial association may be easily missed. To our knowledge, this is the first report of a family with multiple cases of NSCLC associated with germline transmission of an EGFR V834L germline variant; all tumors in the four family members additionally harbored a somatic EGFR L858R mutation in cis. All except one of the known V834L carriers developed lung cancer during their lifespan, but so far none of the investigated noncarriers.
There are only a few reports documenting families with proven inherited EGFR variants over generations that probably conferred predisposition to lung cancer. 1,2 In a family with EGFR V843I mutations, four family members in two generations (three of them with proven germline variants) developed lung adenocarcinoma. 1 As in the family we studied, all of these adenocarcinomas displayed a somatically-acquired L858R mutation in cis with V843I. The index patient in the family with EGFR V843I mutations was treated with erlotinib and gefitinib, but without response, even though the L858R mutation was present. Also, in vitro, the V843I substitution conferred TKI resistance. Yu et al 2 described a family carrying a germline T790M mutation. In their study, both the index patient and her mother developed lung adenocarcinomas. Interestingly, multiple distinct synchronous tumors of the index patient displayed different additional EGFR mutations (ie, deletions of 3 and 15 bp in exon 19 and L858R). 2 A report by Bell et al 3 describes a family with five members affected by lung cancer. Proven germline EGFR T790M mutations were present in three patients in two generations. 3 In distinct lesions in two of these patients, the mutation co-occurred with different EGFR substitutions (L858R, L747-T751del, and G716A), all in cis with T790M. Because T790M is a well-known TKI resistance mutation, no response was observed upon gefitinib treatment in the only TKI-treated patient of this family, as expected.
Incidental cases of germline EGFR variants in patients with lung cancer have been reported. These have included variants such as V769M, R776H, and T790M substitutions in exon 20 and R831C, V843I, and P848L substitutions in exon 21. [4][5][6][7][8][9] However, in the absence of family histories, it is unclear whether these variants predisposed these patients to lung cancer. Importantly, as described above, these germline variants often co-occur in the tumor with other, mostly well-known, actionable EGFR mutations. This strongly indicates that somatically acquired secondary EGFR mutations are indispensable for tumorigenesis.
We hypothesize that presence of the EGFR V834L germline variant is causative for the high incidence and early onset of lung cancer in the family we studied. So far, all lung cancers in this family have been found to harbor both the EGFR V834L variant and the well-described oncogenic EGFR L858R mutation. Although this is a relatively small cohort, this may indicate that the V834L variant is not or only weakly oncogenic and by itself is not sufficient to drive uncontrolled growth. However, when a second oncogenic mutation in cis is present, cell growth accelerates more than proportionally.
This hypothesis is in line with eight described EGFR V834L variants found in the Catalogue Of Somatic Mutations In Cancer database. 9 The V834L variant in the lung tumors of all of these patients, with unknown germline status, is accompanied by the L858R mutation (seven times) or by an exon 19 deletion. [10][11][12] The reason for the apparent preference of V834L for L858R is unclear. Such a preference is not unique (eg, the germline variant EGFR V769M preferentially associates with mutations of codon G719 and/or S768). This is possibly because the energy balance of the EGFR V769M-substituted protein does not favor combination with L858R. 9 Structural and functional analysis of the V834L variant alone and/or in combination with strong oncogenic driver mutations can provide additional mechanistic information on its pathogenicity and preferred association with other oncogenic EGFR mutations.