Until recently, chromosomal abnormalities leading to pathologic ALK protein expression had been described only in ALCLs, IMTs, and rare diffuse large B cell lymphomas.(14
) The identification of an ALK
-rearrangements in ALCL has traditionally been through karyotypic analysis of metaphase spreads, or FISH analysis of mitotic or interphase nuclei using probes flanking the ALK
) However with the advent of monoclonal antibodies that recognize the ALK protein, IHC analysis of tumor tissues has become a highly sensitive and cost effective surrogate for genetic testing.(24
locus is now recognized to be pathologically dysregulated in approximately 5% of lung adenocarcinomas.(8
) Most commonly, the genetic lesion consists of an intrachromosomal deletion and inversion event resulting in an EML4-ALK
fusion that cannot be detected by conventional karyotypic analysis.(8
) Therefore, the diagnosis of ALK
-rearranged lung adenocarcinoma requires IHC, FISH, or RT-PCR based analysis of biopsy tissue.
Currently several laboratories rely on FISH analysis of mitotic or interphase tumor nuclei and identify a “split” hybridization signal to establish the presence of an ALK
) However FISH is unlikely to be a preferred method for screening lung adenocarcinomas in routine surgical pathology practice because 1) the “break-apart” signal pattern resulting from the intrachromosomal deletion and inversion event in the setting of polysomy typical of lung cancer is subtle and easily missed (i.e. compare with ), 2) morphologic indicators of tumor versus non-neoplastic stromal or normal epithelial tissue is lost with FISH analysis, and 3) FISH remains a specialized test not routinely performed by many pathology laboratories. We previously highlighted the potential difficulty of this test when we reported a case of lung adenocarcinoma that was originally classified as ALK
-germline due to mis-interpretation of the FISH analysis (16
). The error was recognized upon finding positive staining for ALK-protein expression by IHC, and the original FISH analysis was re-reviewed and found to be in error.
Alternatively, some laboratories have used a multiplexed RT-PCR based assay to detect EML4-ALK
or alternative fusion transcripts.(28
) However this technique is also specialized, not performed in many routine pathology laboratories, and the vast majority of specimens submitted for histologic diagnosis are stored as FFPE tissue in which the RNA may be substantially degraded. Furthermore, this technique may not be able to detect all the translocations involving the ALK
gene, and most importantly, it may not be entirely specific.(17
) Never the less, we had sufficient tissue to perform RT-PCR analysis on 10 cases determined to have an ALK
rearrangement by FISH. Each of these cases was positive for ALK-protein by IHC using the D5F3 antibody, however in only 9 cases were we able to detect an ALK fusion transcript by RT-PCR (Table S1
). Our failure to detect the ALK fusion in one case may have been due to the presence of an alternative EML4-ALK fusion variant or an ALK
fusion with a gene other than EML4.
IHC remains a preferred technique for screening and diagnosis in routine surgical pathology practice and could prove to be a fast and cost-effective method to identify NSCLC patients for clinical studies of ALK inhibitors. We, and others, have shown that IHC-based screening can be used to identify lung adenocarcinomas harboring known or novel ALK
) In an analysis of 10 cases of ALK
-rearranged lung adenocarcinoma, we previously reported that a conventional, diagnostic IHC test to detect ALK
-rearrangements in ALCL detected ALK protein expression in only 4 of 10 cases of ALK
-rearranged lung adenocarcinoma.(16
) We were able to increase the sensitivity of the test and provide evidence of ALK protein expression in 4 of the remaining 8 cases by including a non-traditional tyramide-based amplification step in our protocol. Others have published similar results using a different, non-traditional amplification technique.(30
) However it remained unresolved whether those cases with an ALK
-rearrangement detected by FISH but negative for ALK expression by IHC express the mutant protein at all.(32
Using 22 ALK
-rearranged lung adenocarcinomas, among the largest collections reported to date, and 19 ALCLs, we compared ALK protein expression by the quantitative IHC analysis using one well-described antibody and two novel antibodies. All three antibodies recognize epitopes within the ALK protein that is preserved in all known ALK
fusions. We find that tumor specific ALK protein expression in ALK
-rearranged lung adenocarcinomas is much lower than that in ALK
-rearranged ALCLs, and that this low level of protein expression necessitates higher titers of antibody for IHC-based detection. Furthermore we found that IHC using the novel antibody D5F3 shows much greater sensitivity than the antibody ALK1 in detecting ALK
-rearranged lung adenocarcinoma by either objective image analysis or by 3 pathologists' individual interpretations. Most importantly, the interpretation of this novel IHC test is highly reproducible among pathologists and shows complete concordance with genetic data. We acknowledge that in addition to ALK1, D5F3, and D9E4, there are other commercially available antibodies that can detect ALK protein expression in ALCL.(30
) However, due to our limited tissue available from ALK
-rearranged lung adenocarcinomas, we could not extend our study with additional antibodies. Similarly, we were unable to perform a correlative study between EML4-ALK
transcript expression and ALK protein expression as detected with our novel antibodies. As ALK
-rearranged lung adenocarcinomas become more easily identified and larger collections are assembled, these additional comparisons can be made.
It has become clear that ALK
-rearranged lung adenocarcinomas are a rare tumor with unique clinico-pathologic characteristics. Patients with ALK
-rearranged lung adenocarcinomas are unresponsive to TKIs that target EGFR, however a novel TKI that targets ALK have shown dramatic clinical responses in an ongoing clinical trial.(12
) Therefore the accurate and timely identification of patients with ALK
-rearranged lung adenocarcinomas is likely to be of therapeutic importance. We believe that IHC using the novel rabbit monoclonal antibody D5F3 is a preferred method for identifying ALK
-rearranged lung adenocarcinomas in routine clinical practice.
Statement of Translational Relevance
Approximately 5% of lung adenocarcinomas harbor the EML4-ALK gene fusion and emerging clinical data indicate that these tumors may be responsive to inhibitors that target ALK. However it remains unclear whether all lung adenocarcinomas harboring an ALK rearrangement express mutant ALK protein. We demonstrate that ALK-rearranged lung adenocarcinomas invariably express ALK protein, but at much lower levels than in anaplastic large cell lymphoma. As a result, more than 30% of ALK-rearranged lung adenocarcinomas are not identified by standard immunohistochemistry (IHC) assays. Using a novel monoclonal antibody with increased sensitivity for ALK, we have developed an IHC assay that accurately identifies ALK-rearranged lung adenocarcinoma with high reproducibility, sensitivity and specificity. This assay will facilitate the routine identification of ALK-rearranged lung adenocarcinomas in clinical practice and detect lung cancers that may be responsive to ALK inhibitors.