Clonality Analysis for the Relationship between the Pulmonary Combined Neuroendocrine Carcinoma and "the So-Called Reported Histologic Transformation"

Abstract

Histologic transformation (HT) is common following targeted therapy in adenocarcinoma. However, whether the transformed tumor is a new component or a combined neuroendocrine carcinoma (C-NEC) remains controversial. We aimed to explore the relationship between pulmonary C-NEC and HT. Macro-dissection was performed on different components of surgically resected C-NEC samples. Molecular alterations and clonal evolution were analyzed using whole exome sequencing (WES). The gene statuses for TP53 and RB1 were determined using immunohistochemistry (IHC) and WES to analyze the relationship between C-NEC and reported HT. Sixteen combined small-cell lung cancer patients and five combined large-cell neuroendocrine carcinoma patients were enrolled. The frequency of p53 and Rb inactivation, assessed using IHC in NEC and non-NEC components, was 76.2/76.2% and 66.7/61.9%, respectively. The expression consistency between the components was 81.0 and 85.7% for p53 and Rb, respectively. The frequencies of TP53, RB1, and EGFR mutations, assessed using WES in NEC and non-NEC components, were 81.0/81.0%, 28.6/28.6%, and 42.9/42.9%, respectively. The concordance rates for TP53, RB1, and EGFR were 90.5, 71.4, and 90.5%, respectively. The consistency rate between IHC and WES was 81.0 and 61.9% for TP53 and RB1, respectively. The different components had a common clonal origin for the 21 C-NECs in the clonal analysis, consistent with previous studies on HT. Our study shows that IHC is more sensitive for Rb detection and C-NEC, and the reported HT may be due to differences in evaluations between pathologist and clinicians. Assessing the p53/Rb and EGFR status for such cases would help in recognizing potential transformation cases or uncovering potential combined components.

Keywords: RB1; clonality analysis; combined neuroendocrine carcinoma; histologic transformation; immunohistochemistry; whole exome sequencing.

Figure 1

Case T21, which was initially diagnosed as ADC in an outside organization, predominantly had an acinar growth pattern with a partially with solid structure (A). After taking oxitinib orally for one year, the patient’s PET-CT and chest CT showed a hypermetabolic focus and abnormal mass under the right diaphragm and invasion to the diaphragm, right lung parenchyma, and liver tissue (B). Then, the patient received a partial hepatectomy with a partial right lower lobe and partial diaphragmactomy. Postoperative pathological HE images are shows in (C), which were diagnosed as SCLC. The immunohistochemistry result is exhibited in (D), with CK, TTF-1, CD56, and Syn positive for SCLC. The Ki-67 index was approximately 70%. Due to the inconsistent diagnosis, the pathologist supplemented the remaining tumor tissue. Only a small-sized ADC component was observed in the diaphragm tissue (E) before, (F)). Similarly, the tissue after manual dissection was stained with HE (E) after. Also, the ADC component was confirmed by NapsinA (exhibited in a higher magnification in the lower right (40×) and TTF-1 (G)). p53 was 95% diffuse and strongly positive, and Rb was negative in both components. The X-ray images showed postoperative changes after resection of the lung and diaphragm (H).

Figure 2

Common driver gene analysis in 20 C-NEC (except case T21). The C-NEC was classified as SCLC/ADC, SCLC/SCC, LCNEC/ADC, LCNEC/SCC, forming four groups. TP53, EGFR, and RB1 were the most common molecular alterations.

Figure 3

Representative clonal relationship between two tumor components of combined SCLC/ADC, LCNEC/ADC, LCNEC/SCC, and SCLC/SCC patients shown in a fish plot and a two-dimensional plot. (A–D) Representative patients who met criteria (i), in which possibly damaging clonal driver mutations were shared by two tumor components. (E) Representative patient who met criteria (ii), in which possibly damaging driver mutations were clonal in one tumor component but subclonal in another paired tumor component. (F) Representative patient who met criteria (iii), in which ≥2 clusters with passenger mutations were shared by two tumor components and were clonal in at least one tumor component. T0: default virtual point by software, CCF: cancer cell fraction.

Figure 4

Clonal relationship between three tumor components of a combined SCLC/ADC patient shown in a three-dimensional plot and a fish plot. (A,B) Patient T21 who met criteria (iii), in which ≥2 clusters with passenger mutations were shared by three tumor components and were clonal in at least one tumor component.