ALK Expression in Rhabdomyosarcomas: Correlation with Histologic Subtype and Fusion Status

Abstract

Immunohistochemical staining for anaplastic lymphoma kinase (ALK) has been described in rhabdomyosarcomas (RMS), especially the alveolar subtype. Previous studies have yielded conflicting results regarding the pattern of staining (nuclear versus cytoplasmic), and there has been no correlation with PAX3-7/FKHR fusion status. This study was undertaken to evaluate ALK receptor protein expression in a large series of RMS; to correlate these results with fusion status; and to investigate the possibility of 2p23 amplification or translocation using fluorescence in situ hybridization (FISH). Sixty-nine cases of RMS were examined and classified as alveolar RMS (ARMS), embryonal RMS (ERMS), or unclassifiable RMS (URMS) subtypes. Anaplastic lymphoma kinase immunohistochemistry was performed using anti-human CD246 antibody; cases were considered positive when more than 50% of cells had moderate or intense cytoplasmic and/or nuclear staining. There were 30 ARMS, 37 ERMS, and 2 URMS subtypes. Reverse transcription-polymerase chain reaction for PAX3/PAX7-FKHR fusion analysis had been done in all cases of ARMS, in 27 of 37 cases of ERMS, and in both URMS cases. Anaplastic lymphoma kinase staining was positive in 16 of 30 ARMS (53%) and 9 of 39 nonalveolar RMS (23%) cases (P < 0.05). Of the 21 ARMS cases with PAX3-FKHR fusion, 10 of 21 (48%) were positive for ALK staining; of the 6 ARMS cases with PAX7-FKHR fusion, 3 of 6 (50%) were positive for ALK staining; and 3 of 3 (100%) of the fusion-negative ARMS were positive with ALK staining. When comparing each of the ARMS subtypes, statistical significance was not reached. All positive cases showed dot-like cytoplasmic staining; nuclear staining was not seen. Of a subset of 6 ALK-positive ARMS submitted for break-apart FISH for the ALK locus, there was no evidence of a translocation; 1 case had ALK amplification and 2 had low-level gains of the ALK gene. We conclude that there is ALK overexpression in RMS, more commonly in ARMS than in ERMS, most likely independent of fusion status. Amplification or upregulation of ALK may underlie ALK protein overexpression.

Anaplastic lymphoma kinase status in rhabdomyosarcomas

“…Further, the regulatory sequences of the ALK gene were shown to contain a high-affinity binding site to the PAX3-FOXO1 protein. 38 Although a previous study 20 found no association between FOXO1 rearrangement and ALK immunoreactivity, this discordance may be owing to the difference in the number of cases studied, the ALK staining protocol used, and the molecular method employed to determine the gene rearrangement status.…”

“…Other investigators also reported ALK gene gain/amplification in rhabdomyosarcoma (Table 4), but the number of cases studied was small, and the quantitative definition of gain/amplification was not provided. 19,20,22 Notably, these prior studies either did not use a reference probe 19,22 or separately examined the ALK gene and a reference gene, 20,23 whereas we simultaneously evaluated the ALK and reference genes using a dual-color approach that enabled precise determination of gene copy number at the cellular level.…”

“…ALK rearrangement was not identified in any of the cases examined in this study, a finding similar to that of other studies. 20,22,23 Interestingly, one Abbreviations: ABC, avidin-biotin complex; ALK anaplastic lymphoma kinase; ARMS, alveolar rhabdomyosarcoma; ERMS, embryonal rhabdomyosarcoma; HIER, heat-induced epitope retrieval; LSAB, labeled streptavidin biotin; NA, data not available; TRS, targeted retrieval solution (Dako). Cessna et al 19 also separately evaluated p80 antibody, the results of which are not summarized here.…”

“…The ALK inhibitor crizotinib showed a marked overall response and good disease control rates in patients with ALK-rearranged lung cancers, 16 and similar encouraging results were reported for this drug in the case of an inflammatory myofibroblastic tumor 17 and anaplastic large cell lymphomas. 18 Considering several reports that have documented ALK expression in rhabdomyosarcomas, [19][20][21][22][23] we speculated that ALK may also have an oncogenic role in these sarcomas, and that they may be treatable with ALK inhibitors. We first decided to clarify the incidence of ALK expression and genomic changes in rhabdomyosarcomas in relation to clinicopathological parameters.…”

Anaplastic large cell lymphoma: pathology, genetics, and clinical aspects

“…130,131 Alveolar rhabdomyosarcoma and neuroblastoma may also be positive in highly sensitive ALK immunohistochemistry. 70,132 There are generally two types of assays used for fluorescence in situ hybridization analyses of fusion genes, i.e., fusion assays and split (break-apart) assays. In fusion assays, the two genes involved are stained in different colors with the fluorescence-labeled probes.…”