Article Text
PDF
Abstract
In advanced non-small-cell lung carcinomas epidermal growth factor receptor (EGFR) and KRAS testing is often performed on cytology. Liquid-based cytology (LBC), which eliminates the need for slide preparation by clinicians, may be very useful. In 42 LBC DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after smear preparation and laser capture microdissection (LCM) of Papanicolaou-stained cells. EGFR and KRAS mutational analyses were performed by direct sequencing. On CytoLyt-derived DNA four EGFR (9%) and five KRAS (12%) gene mutations were found. When direct sequencing was performed after LCM, the rate of cases that displayed either EGFR or KRAS mutations increased from 21% to 40%. Although time-consuming, LCM makes direct sequencing highly sensitive even on LBC preparations containing only a few cells.
- Cancer research
- colorectal cancer
- cytology
- gall bladder
- lung
- molecular pathology
- oncogenes
- pancreas
- p53
- thyroid
Statistics from Altmetric.com
- Cancer research
- colorectal cancer
- cytology
- gall bladder
- lung
- molecular pathology
- oncogenes
- pancreas
- p53
- thyroid
Lung cancer is the leading cause of worldwide cancer-related deaths. Most cancers are non-small-cell carcinomas (NSCLC).1 In its advanced stages the epidermal growth factor receptor (EGFR) is a major therapeutic target. Activating EGFR mutations, including either in-frame deletions in exon 19 or point mutations in exons 20 (eg, T790M) and 21 (eg, L858R) play an important role in determining responsiveness (exon 19 deletions and L858R mutation) or resistance (T790M) to small molecule tyrosine kinase inhibitors, such as gefinitib and erlotinib. Codons 12 and 13 KRAS mutations may also induce tyrosine kinase inhibitor treatment resistance.1
In advanced NSCLC, histology is often not available and gene testing may only be performed on cytology.2 Various types of cytological preparations have been used. Results on cell blocks, on scraped cells from archival slides and on fresh cells are not entirely consistent.2 A cytopathologist's on-site evaluation of the harvested material, confirming the presence of a sizeable number of neoplastic cells, may be crucial.3 However, in a busy cellular department it may not be possible to provide a cytology team for the assessment of adequacy, because it can take up to 45 min for a single procedure.4 Liquid-based cytology (LBC) makes the processing of material easier and eliminates the need for slide preparation by clinicians.5 To date, only the series published by Boldrini et al6 has included a very small number (n=3) of exfoliative LBC exploited for EGFR testing.
Recently, without laser capture microdissection (LCM) a very low success rate on cell blocks with less than 50% tumour cellularity was reported.7 The hypothesis of this study is that LCM is necessary when LBC is adopted. The aim of our study was to compare the mutation rate obtained by direct sequencing with and without LCM on LBC.
Methods
Cell cultures
DNA from the PC-9 cell line was used as a mutated control for EGFR exon 19, and wild-type control for EGFR exon 21 and KRAS codons 12 and 13. DNA from the H1975 cell line was used as a wild-type control for exon 19 and mutated control for exon 21. The SW1573 cell line was used as mutated control for codon 12 KRAS mutation. A DNA extraction procedure and non-template control were performed, as previously described.8 In order to evaluate the minimal number of cells needed for the detection of the EGFR exon 19 deletion and exon 2 codon 12 KRAS point mutation we laser microdissected 5, 25, 50 and 100 cells from PC-9 (EGFR del) and SW1753 (KRAS mt) cell lines Papanicolaou ThinPrep slides. To assess data consistency, cell line experiments were carried out in triplicate.
Study samples
The molecular cytopathology laboratory at the University Federico II of Naples is a southern Italian large reference centre for treatment-predictive EGFR and KRAS mutation analysis. Between July 2010 and April 2011 a total of 214 NSCLC samples was referred to our laboratory from 12 different southern Italian hospitals. A large portion of cases (105/214) was cytological specimens; these included LBC (n=62), archival slides (n=32) and cell blocks (n=11). We focused on LBC. The study protocol was approved (protocol 185/10) by the University of Napoli Federico II ‘Carlo Romano’ Ethics Committee. Special care was taken to select only those LBC whose phial contained not less than 10 ml of residual CytoLyt (Cytyc UK, Crawley, UK) preservative solution. Forty-two samples were available. Corresponding patient features, obtained by chart review, are reported in table 1. Most (33/42) cases had been diagnosed as adenocarcinoma, two were squamous cell carcinomas and seven were NSCLC not otherwise specified. In eight cases NSCLC subtyping had also been based on thyroid transcription factor 1, p63, cytokeratin 5/6 and Napsin A immunostainings.
Patients (sex, age, smoking history and ethnicity) and sample (site, type and cytological diagnosis) features are correlated to EGFR and KRAS mutational assessments on both cell pellets and LBC samples
LBC processing for EGFR and KRAS gene testing
As standardised operative procedures for EGFR and KRAS analysis on LBC were not available, two different approaches were followed. As shown in figure 1, in each single case DNA was extracted twice. One sample was obtained directly from CytoLyt solution, whereas the other DNA sample was derived after LCM of Papanicolaou ThinPrep slide, as previously described.9 EGFR and KRAS gene testings were performed by direct sequencing, as previously described.9 10
Study design. Epidermal growth factor receptor (EGFR) and KRAS analysis was carried out both on DNA directly extracted from the CytoLyt solution and on that obtained from ThinPrep slide laser microdissected cells. LBC, liquid-based cytology.
Results
Amplification failure was a rare event; all CytoLyt samples yielded adequate results for both EGFR and KRAS sequences. On the other hand, only two cases from microdissected Papanicolaou ThinPrep slides had to be repeated once before obtaining adequate results (data not shown). On CytoLyt-derived DNA four EGFR and five KRAS gene mutations were found. On matched LCM-derived DNA, these mutations were confirmed; however, additional EGFR (E746-A750del n=2; L747-A750del n=1 and I745insKIPVAI n=1) and KRAS (G12D n=2 and G12V n=1) mutations were also found (figure 2). Overall, EGFR and KRAS gene mutations occurred in 19% and in 21% of LBC, respectively.
Electropherograms showing wild-type (1B) and mutated epidermal growth factor receptor (EGFR) exon 19 sequences (1D) resulting, respectively, from CytoLyt (1A) and laser capture microdissection (LCM) (1C) derived DNA. In 1C a Papanicolaou-stained ThinPrep slide is shown before (I) and after (II) LCM. The mutated allele (E746_A750del) sequence is indicated by an arrow in the 1D box. The deleted sequence is 2235_2249delGGAATTAAGAGAAGC.
On cell line Papanicolaou ThinPrep slides EGFR and KRAS mutations were consistently detected on 100, 50, and 25 microdissected cells. Results lacked consistency when only five cells were microdissected. Similarly, on five routine LBC in cases EGFR (n=3) and KRAS (n=2) mutations were detected on only 25 microdissected cells. In most of these cases mutations were only detectable on LCM Papanicolaou ThinPrep slides; in only one instance was the E746-A750 exon 19 deletion also detected on the CytoLyt-derived DNA.
Discussion
Lung cancer cytological samples frequently include a non-neoplastic cell component;2 thus, direct sequencing may lack sensitivity. However, when DNA is selectively obtained from LCM cancer cells, direct sequencing represents the ‘gold standard’.9 In this study, we applied LCM and direct sequencing to LBC. This is significant for several reasons; first, LBC are frequently used in routine practice; for instance, they represented a large portion (62/105; 59%) of the specimens referred to our laboratory for EGFR and KRAS tests. Second, LBC may capitalise the material to enable, besides morphology, DNA and RNA extractions and other ancillary techniques, such as immunostaining that may be required, before EGFR testing, to refine the cytological diagnosis of NSCLC to squamous cell carcinoma or adenocarcinoma. Our series included eight LBC samples that had previously been immunostained by the referring pathologists and were still adequate for EGFR and KRAS genotyping. The rate of EGFR mutation detected in our LBC series (19%) was in line with literature data.11 Higher mutation rates were associated with those cases with adenocarcinoma cytology (24%) and female gender (30%).
This study aimed to evaluate the more appropriate LBC pre-analytical handling. Similar to results previously obtained on histology and conventional smears, problematical samples, in which amplification had to be repeated, were rare.8 12 When direct sequencing was performed after LCM, the rate of cases that displayed either EGFR or KRAS mutations increased from 21% to 40%. Therefore, although time consuming, costly and limited by the requirement of both special instruments and expertise, LCM makes direct sequencing highly sensitive even on a LBC preparation containing only a few cells. In fact, EGFR and KRAS mutations were consistently detected by microdissecting as few as 25 cells on ThinPrep slides from both cell line and routine LBC; this is in line with previous evidence by Molina-Vila et al8 on PC-9 and H1975 cell lines and by Savic et al9 on conventional archivial smears.
In nine cases, in which mutations (EGFR n=4 and KRAS n=5) were detected on the DNA directly extracted from CytoLyt, microscopic evaluation before DNA extraction was not strictly necessary. Therefore, we can foresee that laser microdissection and direct sequencing may soon be replaced by highly sensitive non-sequencing methods on CytoLyt-derived DNA. In this perspective our study may pave the way for improvements. In fact, in this study we first validated LBC for EGFR and KRAS gene mutations and set appropriate LCM and direct sequencing benchmarks against which to evaluate novel approaches. Currently, we are investigating on CytoLyt-derived DNA whether high-resolution melting analysis, for exon 19 EGFR and exon 2 codon 12–13 KRAS, and mutant-specific TaqMan assay, for exons 20 and 21 EGFR, may replace the time-consuming LCM and direct sequencing.
Take-home messages
Liquid based cytology is useful for lung cancer therapy predective tests.
Direct sequencing requires laser capture microdissection of cancer cells from Papanicolaou stained smears.
Molecular techniques more sensitive than direct sequencing could replace direct sequencing enabling testing Cytolyt rather than smears.
Acknowledgments
The H1975 lung tumour cell line was kindly provided by Dr Bianco (Department of Oncology, University of Napoli Federico II). The PC-9 lung tumour cell line was kindly provided by Dr Pallante (IEOS, University of Napoli Federico II).
References
Footnotes
Funding This work was supported by Astrazeneca, grant no ISSIRES0025.
Competing interests None.
Patient consent Obtained.
Ethics approval The study protocol was approved (protocol 185/10) by the University of Napoli Federico II ‘Carlo Romano’ Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.