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. 2016 Sep 13;7(37):58903-58914.
doi: 10.18632/oncotarget.10368.

Anaplastic lymphoma kinase aberrations correlate with metastatic features in pediatric rhabdomyosarcoma

Patrizia Gasparini  1 , Michela Casanova  2 , Raffaella Villa  3 , Paola Collini  4 , Rita Alaggio  5 , Angelica Zin  6 , Paolo Bonvini  6 , Cristina R Antonescu  7 , Renata Boldrini  8 , Roberto Caserini  1 , Massimo Moro  1 , Giovanni Centonze  1 , Cristina Meazza  2 , Maura Massimino  2 , Luca Bergamaschi  2 , Roberto Luksch  2 , Stefano Chiaravalli  2 , Gianni Bisogno  9 , Nadia Zaffaroni  10 , Maria Grazia Daidone  3 , Gabriella Sozzi  1 , Andrea Ferrari  2
Affiliations

Anaplastic lymphoma kinase aberrations correlate with metastatic features in pediatric rhabdomyosarcoma

Patrizia Gasparini et al. Oncotarget. .

Abstract

Rhabdomyosarcoma (RMS) is the most frequent soft tissue tumor in childhood and arises from immature mesenchymal cells committed to skeletal muscle differentiation. Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in several cancers. Moreover, ALK full-length receptor protein has been observed in RMS, although its clinical and functional significance is yet controversial. The role of ALK and its clinical relevance were investigated in a selected cohort of 74 FFPE pediatric RMS and a panel of RMS cell lines, evaluating its gene and protein status, utilizing Fluorescent In Situ Hybridization (FISH), immunohistochemistry (IHC) and Western blot approaches. Moreover, to get insight into its possible therapeutic relevance, effects of ALK silencing on cell proliferation, invasion and apoptosis were studied in RMS cells. ALK IHC positivity was significantly correlated with gene copy number gain, the alveolar subtype, PAX3/7-FOXO1 rearrangements, the presence of metastasis at diagnosis and a worse overall outcome. Furthermore, EML4-ALK fusion gene associated with higher protein expression was identified in an embryonal RMS. ALK silencing in RH30 ALK positive cells strongly inhibited invasion capability. Overall, our data suggest a potential role of ALK in pediatric RMS.

Keywords: EML4-ALK; anaplastic lymphoma kinase; chromosomal rearrangement; metastasis; rhabdomyosarcoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

We declare that there are no potential conflicts of interests in relation to the work described.

Figures

Figure 1
Figure 1. Protein an genetic status of ALK
(A–C) Example of pediatric alveolar rhabdomyosarcoma, RMS 11(A), positive for ALK protein by IHC (B) associated with an acquisition of gene copy number of ALK by FISH (C). (D–F) Example of alveolar rhabdomyosarcoma, RMS 4, (D) with a ALK focal positivity IHC (E), and an acquisition of ALK copy number only in IHC-ALK positive tumor cells (F). (G–I) Example of embryonal rhabdomyosarcoma, RMS 42 (G), ALK IHC negative (H) and disomic with no gene copy number gain by FISH (I).
Figure 2
Figure 2. ALK rearranged ERMS
(A–F) Embryonal rhabdomyosarcoma (RMS 68) with an anaplastic component (arrow) (A)characterized by an EML4-ALK rearrangement. Positivity for ALK protein by IHC is present in the membrane within the anaplastic component as indicated by the black arrow, and within the cytoplams as indicated by the red arrow (B). Positivity for phospho-ALK by IHC is observed in the membrane as well as the cytoplasm (C). FISH analysis utilizing a split apart commercial probe, revealed the presence of an ALK rearrangement as represented by the single red and green signals (as indicated by the arrows; D). EML4-ALK rearrangement was confirmed by FISH, utilizing a EML4-ALK fusion commercial probe. The presence of the fusion yellowish signal (arrows) indicate the fusion gene between the EML4 and ALK gene (E). EML4-ALK fusion transcript as visualized with rt-PCR: lane 1 DNA ladder VI, lane 2 exon 13 of EML4 breakpoint at 155bp and lane 3 exon 20 of ALK breakpoint at 155 bp (F).
Figure 3
Figure 3. Overall survival vs immunohistochemistry
The Kaplan Meier graph clearly shows that RMS with ALK IHC positivity have a worse overall outcome compared to those cases with no protein expression.
Figure 4
Figure 4. ALK silencing: siRNA-mediated down-modulation of ALK significantly inhibited invasion capability of RH30 cells
See this image and copyright information in PMC

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