The Ventana ALK (D5F3) CDx assay (Ventana Medical Systems Inc., Tucson, AZ, USA) has been approved for screening of ALK rearrangement in China and the US since 2012 and 2015, respectively. This standardized IHC assay has been shown to have between 93% and 100% agreement with the results of FISH analysis when used for screening of ALK rearrangement in clinical practice [6, 7].
Cytology specimens play an important role in the diagnosis of lung cancer, especially for inoperable patients, and may be the only tumor material available for diagnosis and molecular marker analysis in up to 44% of cases [11, 12]. The fixative type is an important consideration when using the Ventana ALK (D5F3) CDx assay on cytology specimens, and the one approved by the FDA is formalin-fixed, paraffin-embedded (FFPE) tissue. Studies have shown that alcohol or methanol fixation may cause the loss or a decrease of antigen immunogenicity [16, 18].
Several studies have performed ALK ICC testing on conventional cytology smears and proved the feasibility of the method. In a study by Tanaka et al.[14], 18 cytology smears obtained by bronchoscopic brushing were examined, and the results of ALK ICC analysis on Papanicolau-stained and alcohol-fixed slides were compared with IHC analysis of FFPE tissues. The ICC and IHC results showed a high concordance rate, and the sensitivity and specificity of the ICC test were 85.7% and 100%, respectively [14]. In another study of ALK ICC analysis on direct cytology smears [10], the cytology specimens were fixed in Delaunay solution. The ALK ICC results were correlated with ALK FISH results on the cytology smears, and the sensitivity and specificity of the ICC test were 93.3% and 96.0%, respectively [10].
Since the approval of ThinPrep slides by the US FDA for processing non-gynecological material in 1991, a growing number of laboratories are processing FNA, bronchial brushing, and effusion specimens with this method [19, 20]. In our laboratory, ThinPrep specimens are the only available cytology material for bronchial brushing and imaging-guided FNA specimens. However, there are no published data on the performance of ALK ICC analysis on ThinPrep slides. ThinPrep slides are methanol pre-fixed and alcohol-fixed cytology specimens, which are different to conventional cytology smears. Consequently, this is the first study of the Ventana ALK (D5F3) CDx assay on ThinPrep slides.
The overall prevalence of ALK rearrangement with this method was 13.22% in our study, which is higher than the rates of 3% to 7% reported in previous studies [3,4,5]. We consider that the small sample size takes the major responsibility for this result. However, our results are in line with those of a previous study by our group showing that the prevalence of ALK rearrangement with FISH analysis on cytology specimens was 11.7% [21]. A higher prevalence of ALK rearrangement may be attributed to the presence of advanced disease stages among lung cancer patients who are diagnosed via cytology specimens.
The present study revealed a significant correlation between ALK ICC analysis on ThinPrep slides and ALK PCR/FISH analysis, especially when the semiquantified interpretation system was used. The sensitivity, specificity, PPV, and NPV of the ALK ICC test were 93.75%, 96.97%, 93.75%, and 96.97%, respectively, which are in line with a recent large-scale study of ALK IHC analysis [9]. In this study, 933 FFPE NSCLC tissue specimens were analyzed by both the ALK (D5F3) CDx assay and FISH. The results obtained with the ALK (D5F3) CDx assay were highly concordant with those obtained with the FISH assay, and the PPV and NPV of the ALK (D5F3) CDx assay were 86.0% and 96.3%, respectively.
The results of our study suggest that ALK ICC analysis on ThinPrep slides is a reliable ALK testing method that can be used for identification of patients suitable for treatment with crizotinib, particularly when tissue specimens are not available. An interesting finding was that the binary scoring algorithm recommended by manufacturer did not seem to be as valuable as previously reported [6, 9]. Four ALK-positive specimens that were missed when the binary scoring algorithm was used were positive for ALK rearrangement when the semiquantified interpretation system was used, and all 4 were also positive for ALK rearrangement when tested by PCR. The ICC staining pattern of these 4 cases showed intratumoral heterogeneity and moderate staining intensity, which is not a common phenomenon in ALK IHC based on FFPE tissue in that it often demonstrates diffuse strong tumor tissue staining [6]. As an extreme example, it showed moderate staining in 5% of tumor cells, and faint staining in the other 95% (Fig. 2d, e). However, we believe that the heterogeneity of ALK expression in ICC here is not a real heterogeneous expression. Because in the same sample of PCR detection, their results showed a clear positive, its CT value between 14-20, did not show heterogeneity. As mentioned above, this can mainly be attributed to a loss or decrease of immunogenicity caused by the fixation. For example, in the study of Sauter et al. [18], 13 of the 30 antibodies (43%) in a methanol-fixed cell block failed initial validation using IHC conditions that were established in the study laboratory for FFPE material, but when the IHC protocol was adjusted, immunogenicity was restored for most but not all of the antigens [18]. It is worth conducting a prospective, randomized study that explores the influence of alcohol or methanol fixation in ICC analysis. However,Based on the above data, the semiquantified interpretation system, due to its higher sensitivity, may be preferable for ALK ICC analysis on ThinPrep cytology slides, although there was no statistically significant difference between the 2 scoring systems.
There was one false-positive ALK ICC result in our study. This specimen showed faint staining in 50% of tumor cells and strong staining in another 5%, and was considered positive by both scoring systems (Fig. 2g, h) but negative on RT-PCR. It is well known that PCR analysis of ALK based on several common fusion points (Exon 2, 3, 6, 13, 14, 15, 17, 18 and 20) without including all the variants, leads to a negative result for ALK fusion protein expressed in ICC. Unfortunately, the ALK status of this patient did not have a chance to be confirmed by FISH analysis due to the lack of remaining specimens and non-receipt of treatment with crizotinib.
This is the first report of ALK ICC on ThinPrep slides. Further studies are required to corroborate the results obtained in this study. Furthermore, prospective studies that correlate a positive diagnostic test with patients’ response to treatment are required.