(a) Scanned cell block preparation slide of a lung fine-needle aspiration (FNA). (b) Formalin-fixed paraffin-embedded cell block preparation slide with high cellularity. (c) The inset shows EGFR exon 19 deletion detected by fragment analysis [(a) and (b) H&E staining. (b) Original magnification × 400]
Studies of EGFR somatic mutations using cytological specimens (publications with more than 100 cases)
Patients, n (cytological samples)
Type of cytological samples
Type of preparations
EGFR mutations (%)a
FNA, PLE, PCE, BAL
AS, LBC, CB, FC
TBNA, BB/W, PLE, PCE, FNA, Sputum
Direct seq/Taq man assay/length analysis
FNA, BAL, PLE, PCE
BAL, BW, FNA, PLE
Direct seq/Taq man assay/fragment analysis
DNA can also be extracted from samples with low cellularity by the use of techniques for tumor cell enrichment such as manual microdissection and laser capture microdissection.
ALK Rearrangements in Lung Cancer
A clinicopathologic subset of non-small cell lung carcinomas is characterized by an inversion on the short arm of chromosome 2 that juxtaposes the 5′ end of the echinoderm microtubule-associated protein-like 4 (EML4) gene with the 3′ end of the anaplastic lymphoma kinase (ALK) gene, resulting in a fusion oncogene EML4-ALK . Approximately 5 % of patients with NSCLC have tumors that harbor an ALK rearrangement . Patients with lung carcinomas harboring EML4–ALK fusion oncogene or its variants are eligible to treatment by small-molecule inhibitors that target the ALK tyrosine kinase, specifically crizotinib. Acquired resistance has emerged as the major hurdle in the treatment of ALK-positive NSCLC, because patients typically relapse within 1 to 2 years of starting therapy . More than 90 % of ALK rearrangements involve only three variants; however, up to 20 ALK rearrangements have been described. FISH testing using fluorescently labeled DNA probes to localize the specific genomic regions in the tumor nuclei has been developed and the ALK break-apart FISH set (Abbott Molecular) has received FDA approval. The commercial break-apart probe set binds to areas upstream and downstream of the common rearrangement breakpoint in exon 20 of the ALK gene and includes two differently colored probes: the 5′ probe labeled with green and the 3′ probe labeled in orange fluorochromes, respectively. The 3′ probe is observed as red under the microscope. In non-rearranged cells, the red and green signals are closely located or overlying resulting in a composite yellow (fused) signal. In the setting of the rearrangement, the signals (red and green) separate and splitting of the signals occur. A nucleus is considered positive when displays isolated red and green signals separated by at least 2 signal diameters. An isolated red signal without a concurrent green signal is also considered positive. A single green signal is considered negative. A sample is considered positive if ≥15 % of scored nuclei displays a positive pattern and requires counting a minimum of 50 tumor cell nuclei by a first reader. Additional 50 nuclei scored by a second reader are necessary in cases with >10 % but <50 % positive tumor cell nuclei. An example of a case with ALK rearrangement is illustrated in Fig. 6.2.
(a) Section of a formalin-fixed paraffin-embedded cell block preparation of a pleural effusion. (b) Immunohistochemistry for TTF-1 showing positive nuclei. (c) Immunohistochemistry slide positive for ALK. (D) Interphase fluorescence in situ hybridization (FISH) slide showing cells positive for EML4–ALK rearrangement using break-apart probe [(a) H&E staining. (b) Original magnification × 630. (c) Original magnification × 630. (d) Original magnification × 1,000 original magnification × 1,000]
Other methodologies can be used for the detection of ALK rearrangements but currently the gold standard assay for diagnosing ALK-positive tumors is FISH. Immunohistochemistry for ALK has been proposed and used in Canada and European countries as a screening test before the FISH assay or combined with FISH . An extensive review about the different diagnostic tests as well as the limitations of each test has been recently published .
Other Molecular Alterations
A number of other genomic alterations have been detected in NSCLC, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, ROS1 rearrangement, and c–MET amplifications. Clinical trials have been developed for new agents targeting some of those alterations, but currently there is no need for routine clinical testing of those alterations. Although in some centers KRAS mutations have been routinely analyzed, a recent review about the role of KRAS mutation as prognostic and predictive markers in NSCLC has concluded that targeting KRAS remains experimental, and this oncogene cannot, at this time, be recommended routinely for the selection (or exclusion) of patients for therapy . As targeted therapies become available the recommendations for testing will evolve and change over time. New technologies for high-throughput and multiplex platforms for mutational profiling of tumors including MassArray spectrometry, SNaPshot Multiplex PCR, and next generation sequencing might address the issue of the minimal volume of tissue/cells present in small biopsies and cytological samples, and the need for testing for panels of genes as markers for new-targeted therapies becomes available.