Genomic Characterisation of Small Cell Lung Cancer Patient-Derived Xenografts Generated from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Specimens

<div><p>Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.</p></div

Features of specimen LX104 and its derivative xenograft.

<p><b>A.</b> Diff-Quick stained cytology smear of diagnostic EBUS-TBNA sample. Scale bar = 15 µm. <b>B.</b> Diff-Quick stained cytology smear of experimental EBUS-TBNA sample. Scale bar = 15 µm. <b>C.</b> Haematoxylin and eosin stained section of the diagnostic cell block. Scale bar = 30 µm. <b>D.</b> Diagnostic cell block stained for CD56. Scale bar = 30 µm. <b>E.</b> Haematoxylin and eosin stained section of the derivative xenograft. Scale bar = 300 µm. <b>F.</b> Haematoxylin and eosin stained section of the derivative xenograft. Scale bar = 30 µm. <b>G.</b> Section of the derivative xenograft stained for CD56. Scale bar = 30 µm. <b>H.</b> Section of the derivative xenograft stained for TTF1. Scale bar = 30 µm.</p

Immunohistochemical analysis of EBUS-TBNA diagnostic and research specimens and derivative xenografts.

<p>Scale bar = 30 µm. Blank squares indicate that the sample was not available.</p

Cytopathology and histopathology analysis of EBUS-TBNA diagnostic and research specimens and derivative xenograft.

<p>Scale bar = 30 µm. H&amp;E  =  haemotoxylin and eosin stain. Blank squares indicate that the sample was not available.</p

Patient and sample characteristics.

<p>SCLC  =  small cell lung cancer; N/A  =  not available; H&amp;E  =  section stained with haematoxylin and eosin; Results of cell block sections stained with antibodies against Synaptophysin (Synapto), CD56 and Thyroid Transcription Factor 1 (TTF1) are also shown. Lymph node stations are indicated according to the classification of Mountain &amp; Dressler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106862#pone.0106862-Mountain1" target="_blank">[36]</a>.</p><p>Patient and sample characteristics.</p

Ideogram summarising variants predicted to result in mutations commonly seen in SCLC.

<p>Genes are ranked by % according to their prevalence in the COSMIC database. Mutations detected in both the primary sample and the xenograft are shown.</p

Variants detected in primary samples derivative xenografts.

<p>Variants detected in primary samples derivative xenografts.</p