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Molecular determinations of EGFR and EML4-ALK on a single slide of NSCLC tissue
  1. Paola Ulivi1,
  2. Maurizio Puccetti2,
  3. Laura Capelli1,
  4. Elisa Chiadini1,
  5. Sara Bravaccini1,
  6. Daniele Calistri1,
  7. Wainer Zoli1,
  8. Dino Amadori1,
  9. Piero Candoli3
  1. 1Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST-IRCCS), Meldola, FC, Italy
  2. 2Pathology Unit, S. Maria delle Croci Hospital, Ravenna, Italy
  3. 3Pneumology Unit, Lugo Hospital, Lugo, Italy
  1. Correspondence to Dr Paola Ulivi, Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST-IRCCS), IRST- IRCCS, Via P. Maroncelli 40, Meldola, FC 47014, Italy; p.ulivi{at}


Introduction Tyrosine kinase inhibitors (TKIs) and anti-anaplastic lymphoma kinase (ALK) agents are highly effective for the treatment of non-small cell lung cancer (NSCLC) patients harbouring specific alterations, and molecular characterisation of the tumour is needed even when limited tumour material is available.

Methods 20 patients with a known epidermal growth factor receptor (EGFR) gene status were enrolled: 10 had mutated and 10 had wild type tumours. FISH analysis was performed on one cytological or histological sample to determine EML4-ALK status, after which the same cells scraped off each slide were used to evaluate the EGFR status.

Results In the 10 EGFR mutated patients, molecular analysis showed the same results as those obtained before the FISH test. One patient with an EGFR mutation also showed an EML4-ALK translocation, and both FISH-positive and FISH-negative cells maintained the EGFR mutation.

Conclusions EGFR mutation analysis can be performed on the same sample previously submitted to the EML4-ALK FISH procedure.

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Mutations of the epidermal growth factor receptor (EGFR) gene in patients with non-small cell lung cancer (NSCLC) are predictive of sensitivity to tyrosine kinase inhibitors (TKIs) such as gefitinib. Several studies on NSCLC using gefitinib have reported a significantly higher overall survival in patients whose tumours harbour EGFR gene mutations in exons 18, 19 and 21 than for those with wild type gene tumours.1–3 EGFR mutations occur in about 10%–15% of cases of adenocarcinoma of the lung and are usually mutually exclusive with mutation of the KRAS gene. Exon 19 deletions and L858R point mutations of exon 21 of EGFR are the most frequent alterations (more than 90%) and are associated with the best response to TKIs.4 ,5 All patients with lung adenocarcinoma are thus candidates for gefitinib treatment and are required to undergo molecular analysis of EGFR gene status.

Several new targeted drugs show great promise for the treatment of NSCLC treatment and some are in the process of being introduced into clinical practice. Crizotinib, an orally active small-molecule inhibitor of ALK and c-MET, has been shown to improve overall survival in NSCLC patients with tumours harbouring EML4-ALK translocations, highlighting the importance of knowing whether these alterations are present before treatment decisions are made.6

However, about 70% of NSCLC patients present at first diagnosis with advanced disease,7 which precludes the possibility of surgical treatment and, consequently, the availability of tumour tissue. In such cases, the only material that can be used for molecular analyses is that obtained from biopsy or fine needle aspiration. Several studies have already demonstrated that cytological material is suitable for molecular analysis. In particular, it has been shown that mutational analysis of EGFR and KRAS genes can be performed on fixed and stained cytological slides obtained by trans-oesophageal ultrasound-guided fine needle aspiration, endobronchial ultrasound fine needle aspiration and also by other bronchoscopic procedures.8–11 Moreover, it has been seen that FISH analysis of EML4-ALK translocations can be successfully performed on cytological samples.12

One of the main problems in using cytological samples for molecular analysis is that this is usually the only diagnostic material available to the pathologist. Although it has been shown that EGFR molecular analysis can be performed on only a few cells scraped off a cytology slide, permitting the remaining material to be archived, a problem may arise when both EGFR and EML4-ALK analyses are required.

Results obtained in our laboratory have highlighted that molecular analysis of EGFR is not influenced by previous staining with Papanicolau, Fast Quick Rapid, May-Grunwald-Giemsa or H&E8 and that it can thus be performed on tumour material previously stained for diagnostic purposes. In the present study, we aimed to verify whether FISH analysis of EML4-ALK interferes with subsequent EGFR molecular analysis or whether both analyses are possible on the same slide.


We selected 20 patients whose tumours had previously been characterised for EGFR status on paraffin-embedded tissue sections or cytological smears: 10 had an EGFR mutation and 10 had wild type EGFR gene. Another histological/cytological section or cytological smear was selected for each patient from archived material and submitted to EML4-ALK FISH analysis. For this purpose, the selected formalin-fixed and paraffin-embedded histological or cytological sections and cytological samples were treated according to the manufacturer's instructions (Abbott/Vysis, Illinois, USA); FISH hybridisation was performed using the Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe. In view of the risk of detachment of biological material, Superfrost slides (Bio-Optica, Milan, Italy) were used to guarantee perfect adhesion of cytological material. Moreover, each slide was processed individually to exclude the possibility of cross contamination among cytological samples during all the procedures (including pretreatment and washing steps). Samples were deemed to be FISH-positive if more than 15% of scored tumour cells had split ALK 5′ and 3′ probe signals or had isolated 3′ signals. Slides were evaluated independently by two experts blinded to the patient's history and histological findings.

After microscopic evaluation of FISH results, the coverslip was removed from the slides and tumour cells were scraped off and submitted to overnight DNA extraction in a lysis buffer containing 50 mM of KCl, 10 mM of Tris-Hcl pH 8.0, 2.5 mM of MgCl2 and Tween-20 (0.45%) in the presence of 1.25 mg/ml of proteinase K. EGFR mutation analysis was then performed by pyrosequencing methodology, using the EGFR TKI response (sensitivity) kit (Diatech Pharmacogenetics). The procedure we used is shown in figure 1.

Figure 1

Schematic procedure of the analyses starting from a histological (A) or cytological (B) sample. Samples were first submitted to EML4-ALK FISH analysis after which cells from the same samples were scraped off the slides and analysed for epidermal growth factor receptor mutation.


In the 10 patients whose tumours harboured EGFR gene mutations, molecular analysis showed the same results as those obtained before the FISH test: 7 exon 19 deletions and 3 L858R point mutations. Moreover, one patient with an EGFR mutation (exon 19 del E746-A750) also showed an EML4-ALK translocation detected in 37% of tumour cells in the histological sample and confirmed in a cytological sample derived from the same lesion. After FISH determination we scraped off about 40–50 FISH-positive and 40–50 FISH-negative cells from the slide, under the microscope, and separately submitted them to EGFR analysis to evaluate whether or not EGFR mutation and EML4-ALK translocations were present in the same cells. Mutation analysis revealed that both FISH-positive and FISH-negative cells maintained the EGFR mutation, suggesting that the same cells harboured both alterations. Of the 10 patients with wild type EGFR tumours, mutation analysis performed after the FISH test showed absolute concordance with previous results. None of the 10 patients showed an EML4-ALK translocation.


In our study, EGFR mutation analysis performed after FISH determination was always feasible and always reflected the gene status identified in the first evaluation. Such results would seem to confirm the possibility of performing EGFR mutation analysis by extracting DNA from a cytological or histological sample previously submitted to EML4-ALK FISH analysis. From a practical point of view, this would enable both EGFR mutation and EML4-ALK translocation analyses to be performed in sequence on the same sample, useful when only one cytological slide with few tumour cells is available. It would also result in a different analysis algorithm based on the quantity of biological material at hand. When there is a sufficient amount of material, EGFR mutation analysis should be performed as a first step as EGFR-TKIs are the drugs approved for use in clinical practice. Conversely, in cases where only one cytological slide with a small number of tumour cells is available, EML4-ALK translocation analysis could be performed as the first evaluation followed by the determination of EGFR mutation status on the same cells.

In conclusion, the results obtained could prove clinically useful if we consider the increasing number of promising new agents that require multiple target characterisation in settings in which a limited amount of tumour material is available, as occurs in advanced NSCLC.

Key messages

  • EGFR mutation status can be determined on a sample previously submitted to EML4-ALK FISH analysis.

  • Multiple molecular analysis can also be performed on patients for whom only a cytological diagnostic slide is available.

  • Optimisation of biological samples for molecular analysis is needed because of the increasing number of targeted agents available.


The authors would like to thank Professor Rosella Silvestrini for her invaluable scientific contribution and Gráinne Tierney for editing the manuscript.

View Abstract


  • PU and MP contributed equally.

  • Contributors Conception and design of the work, acquisition of data, or analysis and interpretation of data: PU, MP, LC, EC, SB. Drafting the article or revising it critically for important intellectual content: PU, MP, DC, WZ, DA, PC. Final approval of the version to be published: PU, MP, LC, EC, SB, DC, WZ, DA, PC.

  • Competing interests None.

  • Ethics approval Medical Scientific Committee of IRST-IRCCS.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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