PMC3868006_01
(A) Chest CT scan: apparently single nodule (arrow) with solid and ground-glass components. (B) Low-power and (C, D) higher power microscopic images of H&E-stained section, revealing two geographically-contiguous areas with distinct growth patterns - solid and acinar (6 mm area) and predominantly lepidic (9 mm area). Although this could represent histologic heterogeneity in a single adenocarcinoma, dumbbell-shaped outline of the lesion raised the possibility of collision of two separate primary tumors.
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F1
A 75 year-old woman, 50 pack-year ex-smoker, presented with a lung nodule on a routine follow-up chest CT scan for prior colon adenocarcinoma (Figure 1A). The tumor consisted of two geographically-contiguous areas - a 6 mm area with solid and acinar patterns, and a peripheral 9 mm area with a predominantly lepidic pattern (Figure 1C,D). Although the two areas could represent heterogeneous histology in a single adenocarcinoma, the dumbbell shape of the lesion raised the possibility of a collision of two immediately-adjacent separate tumors. The two areas were dissected out and submitted for EGFR and KRAS molecular testing separately.
Sanger sequencing electropherograms, revealing a KRAS G12A(c.35G>C) mutation in the 6 mm nodule (A) and KRAS L19F(c.57G>T)/T20S(c.58A>T) double mutation in the 9 mm nodule (B). (C) Split-screen view of aligned sequencing reads visualized using the Integrative Genomics Viewer (IGV). Sequencing was performed on a PGM Ion Torrent sequencer and aligned with Ion Reporter software. Alignments of reads at exon 2 of the KRAS gene (displayed here in reverse sequence, corresponding to position 25,398,252 to 25,398,296 of chromosome 12), are represented as gray polygons with bases not matching the reference sequence indicated in color. Individual base mismatches with respect to the reference sequence are displayed in orange for KRAS_35[G>C] and green for KRAS_57-58[CT>AA]. The sequence reads from the 9 mm tumor show that L19F and T20S reside on the same reads and therefore the same KRAS allele.
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F2
Sequencing revealed a KRAS G12A mutation in the 6 mm area, and a novel KRAS L19F/T20S double-mutation in the 9 mm are (Figure 2A). The latter was confirmed to represent same-allele double-mutation - a complex compound substitution (L19_T20>FS) - by next-generation sequencing (Figure 2B), resulting in potential functional cooperativity based on amino acid modeling (Figure 3).
Amino acid modeling of KRAS L19F/T20S (L19_T20>FS) double mutation. L19F and T20S are highly conserved and belong to the core region of KRAS. L19 is predicted to interact with GTP/GDP-binding residues (N116, A146, and K16 that in turn interact with F28) and T20 with the aromatic ring of Y40. Together, bulky substitution of phenylalanine (L19F) and a smaller serine (T20S) likely cooperate to distort the KRAS core in the vicinity of the GTP/GPD binding site thus shifting equilibrium towards the active "ON" state.
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F3
The latter was confirmed to represent same-allele double-mutation - a complex compound substitution (L19_T20>FS) - by next-generation sequencing (Figure 2B), resulting in potential functional cooperativity based on amino acid modeling (Figure 3).