Concordance of anaplastic lymphoma kinase (ALK) gene rearrangements between circulating tumor cells and tumor in non-small cell lung cancer

Abstract

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

Keywords: ALK-gene rearrangement; circulating tumor cells; fluorescent in-situ hybridization; lung cancer; molecular diagnosis.

Figure 1. Representative appearance of NSCLC adenocarcinoma with signet ring cells features

A. H&E stain showing solid nests of tumor cells B. Solid with signet ring cells (arrow ) C. Thyroid transcription factor-1 (TTF-1) IHC stain showing strong nuclear reaction in the signet ring cells. D. Solid tumor showing focal positive reaction for (PAS-D) within mucin vacuoles (arrow).

Figure 2. High concordance of ALK FISH rearrangements patterns between CTCs and tumors in NSCLC adenocarcinoma patients

A. Representative ALK FISH rearrangement patterns in CTCs and tumors showing 1F1R1G rearrangement patterns. Yellow, red and green arrows represent fusion (F), orange (R) and green (G) fluorescent signals. B. Representative vimentin (upper panel) and E-cadherin (lower panel) IHC in tumor and control bronchiolar epithelium (black arrow).

Figure 3. Number of cells with ALK rearrangements in ALK-positive NSCLC patients is significantly higher compared to ALK-negative and healthy donors

Graph represents statistical analyses of the data on Table S1 using the non-parametric two-tailed t-test. NS represents not significant while p value <0.05 were considered significant.

Figure 4. An index case suggests that ALK-rearranged CTCs could have clinical application as a diagnostic biomarker to monitor crizotinib treatment and response

A. CT scan taken at baseline, partial response and progression time points showing presence of metastatic tumor in liver (arrow). CTC counts and ALK rearrangement patterns for each time point is indicated in the lower panel. B. Representative images showing 1F1R1G, 2R2G and 1F1R ALK rearrangement patterns following progression on crizotinib treatment. Yellow, red and green arrows represent fusion (F), orange (R) and green (G) fluorescent signals.