Diphenylterazine br In order to gain more insights into the efficacy

In order to gain more insights into the efficacy of TKI on rare and Diphenylterazine EGFR mutations, we screened our in-house data-base from the last 7 years for these mutations (excluding cases with the secondary resistance mutation p.T790M). Within the 408 EGFR-mutated samples in our cohort, 41 samples contained a rare EGFR mutation with unknown or uncertain effect on TKI sensi-tivity. An additional 22 samples had compound mutations with at least one rare EGFR mutation. For these 63 samples, information about the clinical outcome of the affected patients was collected. In addition, we interrogated the current status of the mutations in the abovementioned databases and performed a literature search.
Patients and Methods
Mutational Analysis of EGFR Gene
A total of 2906 lung cancer samples were analyzed for EGFR mutations during routine analysis at the Institute of Pathology at the Charité in Berlin between June 2010 and June 2017.
Sanger DNA sequencing of exons 18, 19, and 21 of the EGFR gene was used until 2014. Sequences of the primer used for PCR
Table 1 Types of Rare EGFR Mutations
Exon Mutation
Numbers in parentheses indicate number of patients with mutation.
Abbreviation: EGFR ¼ epidermal growth factor receptor.
and sequencing are listed in Supplemental Table 1 in the online version. From 2014, next-generation sequencing was used, also including exon 20 in the analysis. Libraries for next-generation sequencing were prepared with the Ion AmpliSeq Colon and Lung Cancer or Ion AmpliSeq Cancer Hotspot Panel (Life Tech-
nologies). Sequencing was performed on an Ion Torrent PGM or S5-XL device. All methods have been described elsewhere.17,18  Juliane Martin et al
Table 2 Types of Compound Mutations in EGFR Gene (Excluding T790M Mutation)
Mutation 1 Mutation 2
Numbers in parentheses indicate numbers of patients with mutations.
Abbreviation: EGFR ¼ epidermal growth factor receptor. aCommon tyrosine kinase inhibitoresensitive mutations.
the routine diagnostic archive. All patients had provided consent for their tissue specimens to be used for research purposes.
Results
EGFR Mutations Detected in Lung Cancer Samples
In total, in 1.4% of all 2906 analyzed lung cancer samples, a rare mutation and in 0.8% a compound mutation (excluding samples with an acquired p.T790M mutation) were detected (Figure 1).
Patient Characteristics
All available clinicopathologic patient characteristics are listed in Supplemental Tables 2 and 3 in the online version, including pa-tient age and sex, histology of the cancer sample, tissue distribution, disease stage, and information about possible medication received before EGFR analysis. Our retrospective study relied on tissue from  Characteristics of Rare and Compound EGFR Mutations
Overall, we detected 36 different rare single mutations in 41 samples. The mutation p.L747P was detected twice, and 5 different samples harbored the mutation p.A767_V769dup (Table 1).
For the 22 cases of compound mutations, a total of 44 mutations (2 mutations per case) were detected, but not all of these were rare
Rare and Compound EGFR Mutations
Table 3 Characteristics of Patients With Rare Mutations (N [ 41 or 40a)
Characteristic Value
Sex
Median
Histology
Squamous-cell carcinoma 1 (2.4)
Data are presented as n (%) unless otherwise indicated. aFor one sample, age of patient was not reported.
(Table 2). TKI-sensitizing mutations were present together with a rare mutation in 16 samples (7 p.G719X, 4 exon 19 deletions, and 5 p.L858R mutations). Additionally, some rare mutations were detected in 2 (p.N700S, p.L833_V834delinsFL and p.L861R) or in 3 samples (p.S768I, p.L861Q). The rare mutation p.T725M was detected as a single mutation in one patient and in combination with a second mutation p.K728E in another patient. Thus, another 20 rare EGFR mutations were detected in the 22 patients with compound mutations.