Chen W et al. (2020), Identification of anaplastic lymphoma kinase fu...

ECB-ART-49882

Oncol Rep 2020 Mar 01;433:817-826. doi: 10.3892/or.2020.7462.

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Identification of anaplastic lymphoma kinase fusions in clear cell renal cell carcinoma.

Chen W , Li W , Bai B , Wei H .

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As one of the most common types of renal cancer, clear cell renal cell carcinoma (ccRCC) in advanced stages constitutes a continued major challenge for uro‑oncologists, as the identification of novel driver mutations and the development of novel targeted therapies against them remain an unmet need. Aberrations in anaplastic lymphoma kinase (ALK), a rational therapeutic target, as verified in lung cancer with ALK rearrangement, have been implicated in the pathogenesis of multiple human cancers. In the present study, we screened ALK expression in 87 pathologically defined ccRCCs via immunohistochemistry (IHC) using a newly developed rabbit anti‑human ALK monoclonal antibody (clone D5F3). Four patients tested positive for ALK expression, as confirmed by IHC. Among them, 2 patients were further confirmed with fluorescence in situ hybridization (FISH) assay with the use of the Vysis LSI ALK dual color break‑apart probe. Furthermore, we detected the existence of the echinoderm microtubule‑associated protein‑like 4/anaplastic lymphoma kinase (EML4‑ALK) (E13:A20, variant 1) fusion gene in tumors from these two patients by using rapid amplification of cDNA ends (RACE)‑coupled PCR sequencing and RT‑PCR. Notably, we first showed that enforced EML4‑ALK expression could significantly promote in vitro proliferation, clonogenic colony formation and apoptosis resistance in HK2 immortalized normal renal tubal epithelial cells and their in vivo outgrowth when injected into immunocompromised nude mice. Importantly, this pro‑tumorigenic effect was completely abolished by the ALK‑specific inhibitor crizotinib, indicating the potential effectiveness of ALK‑specific inhibitors in treating ALK‑rearranged ccRCC patients. Our data revealed that ALK fusions exist in adult ccRCC, providing a rationale for ALK inhibitor therapy in selected patients with ccRCC.

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	Figure 1. Photomicrographs of ALK-germline and ALK-rearranged lung adenocarcinomas and normal adult human renal tissues stained with the D5F3 antibody. (A) ALK-germline sample. Scale bar, 50 µm. (B) ALK-rearranged sample. Scale bar, 50 µm. (C) Normal adult human renal tissues. Scale bar, 100 µm. ALK. anaplastic lymphoma kinase.
	Figure 2. Photomicrographs of ccRCC tissues stained with the D5F3 antibody showing positive expression. Sample 416 (A and E) sample 413 (B and F) sample 405 (C and G) and sample 384 (D and H). (A-D and E-H) Haematoxylin and eosin (H&E) staining and ALK staining, respectively. Scale bar, 20 µm. ccRCC, clear cell renal cell carcinoma; ALK, anaplastic lymphoma kinase.
	Figure 3. Identification of the 5′ ALK fusion partner and EML4-ALK expression. (A) Electrophoresis of products from the RT-PCR analysis of tumor cDNA with the EML4 exon 2 forward and ALK exon 20 reverse primers shows an expected 1,399-bp product (column 3, sample 405; column 4, sample 413). In column 2, cDNA from H2228 lung cancer cells containing a known EML4-ALK fusion gene (variant 3) was amplified by the same primers, which showed an expected 599-bp product. In columns 5 and 6, samples 565 and 568, with negative ALK expression by IHC, were included as the negative control. In columns 8–12, GAPDH was amplified as a positive control (250 bp), and in columns 3 and 7, a negative control reaction without cDNA was run. In columns 1 and 14, DNA molecular markers are shown. (B) Sequencing results from samples 405 (upper panel) and 413 (lower panel) show the sequence of the EML4-ALK fusion breakpoint; the DNA base on EML4 exon 13 (blue) is immediately followed by the DNA base on ALK exon 20 (pink). (C) FISH analysis with the Vysis LSI ALK dual colour break-apart probe confirmed the presence of ALK rearrangement. The white arrow denotes split red-green signals indicative of ALK rearrangement, while touching red-green signals are not indicative of an ALK rearrangement. EML4-ALK, echinoderm microtubule-associated protein-like 4/anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization.
	Figure 4. Histological and immunohistochemical analyses of ALK-rearranged cases. (A) Sample 405; this patient's tumor tissue presents gland ducts and an acinar arrangement, cuboidal cells, a translucent cytoplasm, a round nucleus, nucleolus partially visible under low magnification, and blood sinus dilatation and congestion. (B) Sample 413; this patient tumor tissue shows a group-like arrangement with rounded and polygonal cancer cells, a translucent cytoplasm, a small round nucleus and vaguely visible nucleoli, and rich vascularity in the interstitial space. These two neoplasms were positive for carbonic anhydrase 9 (CAIX), HLA class I (ABC) and cluster of differentiation 10 (CD10). ALK, anaplastic lymphoma kinase.
	Figure 5. EML4-ALK overexpression has a protumorigenic effect on normal renal epithelial cells. (A) EML4-ALK expression in HK2 cells stably infected with a control or EML4-ALK-carrying retrovirus determined by western blot analysis. In vitro (B) proliferation and (C) clonogenic colony formation assays. HK2 cells stably expressing the control or EML4-ALK retrovirus were plated into 6-well plates and cultured for 10 or 12 days, and cell counts were performed every 3 days (B) and colonies were imaged (C, left) and counted at the end of the experiment (C, right). The data are expressed as the mean ± SD of triplicate samples. (D) HK2 cells stably expressing the control or EML4-ALK retrovirus were plated into 6-well plates with (untreated) or without serum (serum starved) for 24 h, and cell apoptosis was analyzed by Annexin V/PI staining and flow cytometry. The data are expressed as the mean ± SD of triplicate samples. (E) Tumors were established by subcutaneously injecting HK2 cells stably expressing the control or EML4-ALK retrovirus into the right flanks of nude mice. When the xenograft reached 100–150 mm3 (control HK2) or 250–350 mm3 (EML4-ALK HK2), animals were randomly divided into two groups and intratumorally injected with vehicle (DMSO) or crizotinib (250 mg/kg) twice weekly for 2 weeks. Tumor formation was monitored, harvested tumors were imaged (E, left), and their volume was calculated (E, right). Each time point represents the mean tumor volume for each group. Data are representative of 2–3 independent experiments. P<0.05, P<0.01, **P<0.001, paired Student's t-test (for B, C and D) or two-way ANOVA with multiple comparisons corrected with the Bonferroni method (for E). EML4-ALK, echinoderm microtubule-associated protein-like 4/anaplastic lymphoma kinase.

References [+] :

Butrynski, Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor. 2010, Pubmed