Comprehensive Molecular and Clinicopathologic Analysis of 200 Pulmonary Invasive Mucinous Adenocarcinomas Identifies Distinct Characteristics of Molecular Subtypes

Abstract

Purpose: Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.

Experimental design: A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).

Results: Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R-NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001).

Conclusions: This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Figure 1.

OncoPrint depicting driver alterations in non-NGS IMA cohort (A). OncoPrint detecting driver alterations and other genetic alterations in NGS IMA cohort (B). Pie chart summarizing the major classes of driver alterations (C) in NGS IMA cohort (n=136). Pie chart depicting the subtypes of KRAS mutations (D) across KRAS-mutant IMAs in both NGS and non-NGS cohort (n=151). ^, KRAS mutations not identified by MALDI-TOF MS and only detected by high sensitivity Sanger sequencing with LNA probes. *, fusion genes not identified by MSK-IMPACT and only detected by MSK-Fusion. #, the case harboring concurrent ERBB3 G284R and D581N mutations. +, the case with NRG2 fusion detected by whole transcriptome sequencing. ~, the case with STK11 truncating fusion detected by whole transcriptome sequencing.

Figure 2.

Histologic findings in IMAs with NRG1/2 fusions. (A) This NRG1-rearranged carcinoma shows classic mucinous morphology with tall columnar cells, basally-located nuclei and abundant apical cytoplasmic mucin. (B) Necrosis is a common histologic finding in IMAs with NRG1 fusions. (C) A focus of desmoplastic stromal invasion is accompanied by depletion of cytoplasmic mucin. (D) Structural features of NRG1 fusions involving the 5’ and 3’ chromosomal partners identified by MSK-Fusion. (E) This NRG2-rearranged tumor is composed of tall columnar cells with pseudostratified nuclei and abundant cytoplasmic mucin. (F) Schematic illustration of the gene structure and transcript sequence of the F11R-NRG2 fusion product and representation of complementary DNA sequencing reads supporting the fusion transcript by whole transcriptome sequencing.

Figure 3.

Histologic findings in IMAs with ERBB2 alterations. (A) An IMA with ERBB2 fusion shows classic IMA morphology with tall columnar cells containing abundant apical mucin. (B) An IMA with ERBB2 exon 17 insertion mutation shows strips of bland columnar tumor cells in the background of abundant intra-alveolar mucin pools. (C) An IMA with ERBB2 amplification shows bland tumor cells with pyknotic basally-located nuclei and abundant cytoplasmic mucin. (D) Another IMA with ERBB2 amplification shows partially necrotic debris within glandular spaces. (E) Copy number plot with relative (Log₂) tumor/normal ratios (y axis) and corresponding chromosomes (x axis) in the tumor depicted in (D) demonstrating ERBB2 amplification (FC: 40.2), CDKN2A/B deletion (FC: −4.4), and NKX2-1 deletion (FC: −7.0). FC, fold change.

Figure 4.

Comparison of overall survival (A) and recurrence-free survival (B) for IMAs with KRAS mutations, NRG1 fusions, and other driver alterations. The p value shown is for three-way comparison. The p value for KRAS vs NRG1 is 0.014 (overall survival) and <0.0001 (recurrence-free survival).

Figure 5.

Pie chart summarizing the major classes of targetable alterations in IMAs (A) and the best clinical response achieved in patients matched to targeted therapies (B).