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Tumor Discovery CTC characterization for EGFR mutations

sequence artifacts attributed, at least in part, to the low quality The results from mutational profiling of the CTCs are
of DNA, the clonal amplification of DNA strands, and/ generally consistent with the exon 18 – 21 mutational profile
or the need for a relatively high number of PCR cycles due of patients with NSCLC using tumor biopsy. For example,
to the low quantity of starting material, as well as chemical 86.7% of CTC-enriched samples analyzed in this study had
modifications that occur during the NGS workflow. a deletion in exon 19 (E746_A750delELREA), which is
54
However, in this study, CTCs were analyzed for mutations consistent with published literature reporting that around
at a read depth of ×10,000, which has been recommended as 80% – 90% of NSCLC patients with an EGFR mutation have
best practice in the literature. 55,56 Therefore, we believe this either this deletion or an exon 21 L858R mutation. 2,8,12,28
reduces the probability of methodological errors. In contrast, However, the frequency of mixed mutations was much
some studies have suggested that because CTCs comprise a higher in the current study (30%) than that in others, with
very small percentage of the total tumor mass, any mutation Caucasian patients having reported frequencies of 5% – 7%
detected with sufficient coverage should be recognized as for mixed mutations. 56-58 The difference in frequency rates
a potentially important clinical variant. The incidence may be attributed to the relatively small sample size of the
55
of EGFR mutations using the CTC + NGS matrix will be present study or, as described earlier, the disparity in analysis
investigated in further studies using a microfluidic device techniques. To the best of our knowledge, the only study that
(designed by our group) capable of single-cell RNASeq. has employed NGS for mutational analysis of EGFR mutations
in CTCs obtained from NSCLC reported an incidence of 13%
Table 3. Patients with mixed EGFR mutations
for mixed mutation for a cohort of 37 patients enrolled for the
59
Patient Exon Mutations study, which was higher than that reported in other studies
No. using PCR-based mutation analysis on tumor biopsies but
28 19 Deletion (E746_A750delELREA), P733L was still lower than that observed in the current study. 51,52
30 19 Deletion (E746_A750delELREA), P733L, The higher incidence reported in our study than in the
L841P
study by Marchetti et al. may be due to the difference
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31 19 Deletion (E746_A750delELREA) in isolation technique for CTCs. The latter used CELL
20 R776H SEARCH for isolating CTCs, whereas the present study
32 19 Deletion (E746_A750delELREA), P753S used a novel immunomagnetic microfluidic device. CELL
21 P848L
18 G696E, L703P SEARCH technique has been associated with a CTC yield
40
39 19 Deletion (E746_A750delELREA) of ≤60% and a purity of around 50%. Perhaps the CTCs
20 T790M isolated were not totally representative of the molecular
18 N700D events in the malignant environment, which may have
43 19 Deletion (E746_A750delELREA) resulted in an under reporting of mutations present.
21 V843I Preliminary validation studies on the yield and purity of
17 19 Deletion (E746_A750delELREA) the device in this study using cell lines expressing varying
21 L858R levels of EpCAM spiked in media and blood have shown
40 19 Deletion (E746_A750delELREA) that the device isolates EpCAM-positive cells with a yield
20 L841P, P848L of ≥65% and purity of ≥95% (unpublished data from our
56 20 R766H laboratory). The yield and purity of CTCs isolated using
Abbreviation: EGFR: Epidermal growth factor receptor. The bold values the device may have contributed to the increased mixed
represent mutations/deletions on exons 18-21 of the EGFR gene. mutation incidence reported in the current study.
Table 4. Rare EGFR single nucleotide variants identified in patient CTC‑enriched samples using NGS

Patient ID Nomenclature (SNV) Exon RS no. Amino acid Comments
31,34 c. 2375T˃C 20 132563568 L792P Pathogenic mutation of somatic origin 10,31,60
24 c. 2389T˃A 20 1057519861 C797S Pathogenic mutation of somatic origin associated with drug resistance 10,31,62
31 c. 2327G˃A 20 483352806 R509H Germline origin of uncertain significance 31,60,62
30 c. 2318A˃G 20 121913432 H506R Somatic likely pathogenic 60
37 c. 2123A˃G 18 144932466 K708R Somatic likely pathogenic 63,65
34 c. 2281G˃A 19 121913418 D761N Somatic likely pathogenic 57,62-64
29 c. 2573T˃G 21 121434568 L591R Somatic associated with a drug response 31,60,61
Abbreviations: CTC: Circulating tumor cell; EGFR: Epidermal growth factor receptor; NGS: Next-generation sequencing. The bold values represent
mutations/deletions on exons 18-21 of the EGFR gene.

Volume 3 Issue 4 (2024) 10 doi: 10.36922/td.3987