The proband was a 48-year-old Japanese woman with no previous history of malignant disease (Fig 1; noncritical clinical features have been changed to preserve confidentiality). The patient presented with bilateral multiple pulmonary nodules and metastases to the thyroid glands and multiple bone sites, as well as mediastinal lymph node involvement and pleural dissemination. Pathological examinations resulted in a diagnosis of lung adenocarcinoma, stage IV, T4N2M1. The patient showed no response to several courses of chemotherapy including gefitinib and erlotinib, and eventually died of progressive lung adenocarcinoma. The mother of the proband underwent lobectomy at age 61 years for stage IA lung papillary adenocarcinoma, and is currently alive and disease-free 13 years after the surgery. The aunt of the proband suffered lung cancer at age 70 years, although precise data concerning her disease was unavailable. The younger brother of the proband was diagnosed at age 41 years with stage IA lung papillary adenocarcinoma and underwent lobectomy. He is currently under observation 9 months after surgery. A nephew of the proband suffered non-Hodgkin's lymphoma at age 12 years.

Fig 1.

Peripheral-blood mononuclear cells were collected for the analysis of EGFR mutations from the proband, her younger brother, and her four children. Formalin-fixed paraffin-embedded tumor samples from the proband, her mother, her younger brother, and her nephew, as well as normal lung tissue from her mother and normal intestinal tissue from her nephew, were also subjected to genetic analyses. After receiving approval from the ethics committee of the institutes, written informed consent for the present study was obtained from the proband and all other family members.

A heterozygous EGFR V843I germ-line mutation was identified in the peripheral-blood cells or the normal lung tissue of the three family members with lung adenocarcinoma. In addition, the V843I mutation was also identified in a healthy daughter of the proband. Furthermore, the L858R somatic mutation was also detected in tumor specimens of the three family members with lung adenocarcinoma. Peripheral-blood cells of the three other children and the tumor and the normal tissue of the nephew with lymphoma showed wild-type EGFR in the regions investigated.

To determine whether the somatically acquired secondary L858R mutation occurred in cis or trans to the germ-line V843I mutation, tumor DNA from the three lung adenocarcinoma patients was digested using the restriction enzyme Eae-1, which selectively cuts the wild-type allele at codon 858. Following digestion, the region including codon 843 and 858 was amplified and sequenced. Direct sequencing of all tumor DNA demonstrated a similar level of wild-type valine residue and mutant isoleucine residue signals at codon 843. In contrast, sequence of the DNA digested with Eae-1, which eliminates the wild-type allele at codon 858, revealed that the wild-type valine residue was also greatly reduced at codon 843. These results suggest that the somatic secondary L858R mutation nonrandomly occurred in cis to the germ-line V843I mutation in the three cases.

T790M, another EGFR mutation found in germ-line cells of several families with lung cancer susceptibility, is associated with resistance to tyrosine kinase inhibitors (TKIs), major therapeutic agents of lung cancer.¹ Therefore, we speculated that the V843I mutation might also be associated with TKI resistance. To test this possibility, growth inhibition assay by AG1478, a TKI analog of gefitinib, was applied to the cell line (designated as KCI-PLA1) established from tumor cells obtained from pleural effusion of the proband. The analysis demonstrated that KCI-PLA1 with the L858R and V843I mutations showed resistance to 10 μmol/L of AG1478 (IC₅₀ > 10 μmol/L). In the same assay, the lung adenocarcinoma cell line NCI-H1975 with the L858R and T790M mutations was resistant to 1 μmol/L of AG1478 (IC₅₀ > 1 μmol/L), while the cell line 11-18 with the L858R mutation alone was sensitive to 1 μmol/L of AG1478 (IC₅₀ < 1 μmol/L).

While cases of familial lung cancer have been occasionally reported, their genetic backgrounds remain largely unknown.² The present study showed, to our knowledge for the first time, that the EGFR germ-line mutation is inherited over generations in a family with lung cancer susceptibility. To date, the germ-line V843I mutation has been reported only in a single family with lung cancer susceptibility. Beside this case, five patients with germ-line T790M mutation have been documented within four families with lung cancer susceptibility.^3–6 However, whether the multiple occurrences of lung cancer were related to the inherited EGFR mutation remains unclear, because transmission of the V843I or T790M mutation was not verified in these cases. In contrast, three family members (the proband, her mother, and brother) with lung adenocarcinoma demonstrated identical germ-line V843I mutations together with a somatic L858R mutation in the present series. Our observations therefore indicate that the germ-line V843I mutation is inherited across generations and thereby plays a major role in familial lung adenocarcinoma susceptibility.

The mechanism through which V843I mutation confers predisposition to lung cancer remains unknown. As all of the current cases, as well as the Ikeda et al³ case with lung adenocarcinoma exhibited additional L858R or L861Q mutation in tumor cells, it is possible that the acquisition of a second mutation is essential for the development of lung adenocarcinoma in individuals harboring the V843I germ-line mutation. One possibility is that V843I mutation in the EGFR gene causes genetic instability, thereby predisposing cells for additional mutations within the gene. These mutations may subsequently result in tumorigenesis in cells expressing this mutation. This hypothesis is supported by the finding that the two other sporadic cases carrying the V843I mutation (one non–small-cell lung cancer and one cholangiocarcinoma) also contained a second EGFR mutation.^7,8 Furthermore, the somatically acquired secondary L858R mutation nonrandomly occurred in cis to the germ-line V843I mutation in our study, a finding that also supports the hypothesis.

It is also of note that the proband in the present study, whose adenocarcinoma tissue expressed both the L858R and V843I mutations, showed clinical resistance to gefitinib and erlotinib. Growth inhibitory assays using cell lines established from the patient tumor tissue also showed resistance to TKI, a result that was similar to the TKI-resistant cell line NCI-H1975 that harbored both the L858R and T790M mutations. It is widely accepted that while lung adenocarcinomas with the L858R mutation alone are susceptible to TKI therapy, gain of an additional T790M mutation overrides the effects of the L858R mutation and confers resistance to TKI.¹ Our results suggest that V843I is a TKI-resistant mutation similar to T790M. Both the EGFR T790M and V843I germ-line mutations may be responsible for a unique familial lung cancer syndrome with manifestations of lung cancer susceptibility and resistance to TKIs. The precise molecular mechanisms through which V843I mutation confers predisposition to lung adenocarcinoma and TKI resistance have yet to be clarified.