Combination of conventional immunohistochemistry and qRT-PCR to detect ALK rearrangement

Clinical materials and tissue microarray (TMA) construction

This study included 297 FFPE samples with lung adenocarcinoma diagnosed at the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (CICAMS) in Beijing, between January 2009 and March 2012. Among the 297 cases, 218 were unselected and 79 cases were not effectively treated using conventional treatment.

Among the 218 unselected cases, 178 (with enough tissue) were constructed onto seven TMAs to represent biopsies. A 1.5 mm diameter core was taken from the cancer area based on hematoxylin and eosin (H&E)-stained sections of each sample. The remaining 39 unselected cases (without enough tissue) and 79 selected cases were cut into tissue sections. In the cases where tissue sections/cores fell off the slides during FISH or IHC analysis, tissue sections were re-cut. The collection of these specimens was approved by the National Cancer Center Ethics Committee.

The patients’ medical records were reviewed to obtain their clinicopathological parameters including age at diagnosis, sex, smoking history, tumor size, histological classification and pathological TNM stage.

IHC

Immunohistochemical staining was performed on 4 μm-thick FFPE tissue sections or TMAs. Briefly, the slides were deparaffinized and antigen retrieval was then performed in a steam cooker for 1.5 minutes in 1 mM EDTA, pH 9.0 (Maixin Biological Techology Co. Ltd., Fuzhou, China). ALK (D5F3) rabbit monoclonal (Cell Signaling Technology, Danvers, MA, USA) was applied at 1:150 in SigalStain antibody diluent (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Universal secondary antibody (DAKO) was applied for 15 min. Diaminobenzidine or 3-amino-9-ethylcarbazole was used as chromogens and slides were counterstained with haematoxylin before mounting.

The criteria for scoring ALK were as follows. First, the intensity was graded as 0, negative; 1, weak (light brown); 2, moderate (brown); and 3, strong (dark brown). Second, the proportion of positive tumor cells was graded: 0, no positive cells; 1, <10%; 2, 11%-30%; 3, 31%-50%; 4, 51-70%; and 5, >70%. A final score was derived by adding the two primary scores. Final scores of 0 were defined as “negative expression” (−); scores of 2–5 as “weakly positive expression” (+); and scores of 6–8 as “strongly positive expression” (++).

Fully automated VENTANA ALK (D5F3) IHC analysis was performed as previously described [12]. According to the manufacture’s scoring algorithm, a binary scoring system (positive or negative for ALK status) was adopted to evaluate the staining results. The presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) was considered to be ALK positive while the absence of strong granular cytoplasmic staining in tumor cells was deemed to be ALK negative.

FISH

FISH was performed on 3 μm-thick FFPE tumor tissues using a break-apart probe specific to the ALK locus (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe; Abbott Molecular, Abbott Park, Illinois, USA) according to the manufacturer’s instructions. Tumor cells, the nuclei of which had one or more FISH signals of each color, were enumerated. A positive cell was defined as one in which the nucleus had split signals (two or more signal diameters apart) or a single orange signal (deleted green signal) in addition to fused and/or split signals. A sample was considered positive if >25 cells out of 50 were positive. If a sample had 5 to 25 positive cells (10 to 50%), another 50 tumor cells were counted. If the average percentage of positive cells in 100 tumor cells was <15% (<15/100), the sample was considered negative. If the average percentage of positive cells was ≥15% (≥15/100), the sample was considered positive. TMA cores with high backgrounds or very weak signals that affected the signal assessment were excluded from the analysis.

Real-time quantitative reverse transcription PCR (qRT-PCR)

The EML4-ALK fusion mRNA was detected by qRT-PCR using an AmoyDx EML4-ALK Fusion Gene Detection Kit (Amoy Diagnostics, Xiamen, China). Briefly, total RNA was extracted with an AmoyDx FFPE RNA Kit (Spin Column) from 5–10 μm-thick FFPE sections with over 70% tumor cells. For each sample, 100–500 ng of extracted RNA was used for reverse transcription into cDNA at 42°C for 1 h. Real-time PCR was then carried out in each of the four reactions of the EML4-ALK Fusion Gene Detection Kit according to the manufacturer’s protocol. Reaction 1 amplifies EML4-ALK variants 1, 2, 3a and 3b (variants 1/2/3a/3b); reaction 2 amplifies EML4-ALK variants 4 and 4′; reaction 3 amplifies EML4-ALK variants 5a, 5b, 5′ and 8 (variants 5a/5b/5′/8); and reaction 4 amplifies the reference gene beta-actin. All of the assays were performed on an Agilent Mx3000P QPCR instrument (Agilent Technologies, Santa Clara, CA). The following PCR procedure was used: an initial denaturation at 95°C for 5 min followed by 95°C for 25 s, 64°C for 20 s and 72°C for 20 s to ensure the specificity and 31 cycles of 93°C for 25 s, 60°C for 35 s and 72°C for 20 s to perform the data collection. The quantitative judgment was according to the fusion fluorescence signal. Assay reactions achieving Ct values of ≤30 cycles were considered positive for one of the variants detected by that reaction mixture. A housekeeping gene (beta-actin) was used to control the integrity of the RNA.

Statistical analysis

The statistical analysis of the tumors’ size and age was carried out using Student’s t tests. The values are shown as mean ± SD. The relationship between ALK+ and clinicopathological variables was analyzed with the chi-square test. Statistical significance was defined as p < 0.05.

Concordance of ALK IHC and FISH

Using the newly developed antibody, ALK (D5F3), we analyzed ALK expression in 297 lung adenocarcinoma cases. The cases with strongly or weakly positive ALK expression showed readily appreciable cytoplasmic staining (Figures 1A and 1B). In contrast, the cases with negative expression did not show any discernable staining (Figure 1C). Strong ALK expression was identified in 32 cases, weak expression in 12 cases and no expression in 253 cases (Table 1).

FISH analysis was performed on the 297 cases to evaluate ALK gene rearrangement status. Two hundred and eighty-six out of 297 cases were informative for FISH analysis and 33 cases were identified with ALK+ (Figure 1E). Thirty of the 33 ALK+ cases showed strong ALK expression and the other 3 showed weak ALK expression. Therefore, there were 11 cases that showed ALK expression but were ALK-. We re-reviewed the FISH slides of the 11 discordant cases (2 cases with strong and 9 cases with weak ALK expression), and 3 cases (1 with strong and 2 with weak ALK expression) were identified as ALK+ while 8 (1 case with strong and 7 with weak ALK expression) were still ALK- (Table 1, Figure 1F).

Regarding the 3 ALK+ cases, which were not identified by the original FISH analysis, a case-by-case analysis revealed the following:

Case 1 The dominant FISH signal pattern in this case was more than one copy of a single green signal without a corresponding orange signal in addition to fused signals (Figure 1D). According to the ALK signal enumeration guide, this indicated a deletion of the orange portion of the ALK probe, which targeted the drug targeting area. Therefore, we initially considered this case as negative. After re-reviewing the FISH analysis, we found there were some areas containing scattered ALK+ cells with one or more copies of single green signals in addition to fused signals and a single red signal. The first 50 cells counted revealed 8 ALK+ cells. The second and third cell count in another 100 cells by different readers revealed 6 and 7 ALK+ cells, respectively. If the first and third 50-cell count was considered, the average percentage of positive cells reached 15%. Therefore, this sample should be considered positive.

Case 1 and 3 For these two cases, originally constructed on TMA and IHC, analysis showed strongly positive staining in one core and weakly positive staining in the other. After re-reviewing the FISH slides, we found that there was indeed a small area of each core with a few cells containing subtle break-apart signals. As cell counts were difficult to perform in small areas containing not many cancer cells, we cut the tissue sections. The IHC analysis still demonstrated strongly and weakly positive ALK expression, respectively. The FISH analysis in the tissue sections showed ALK+.

According to the final result of FISH analysis, 36 out of the 286 lung adenocarcinoma cases were identified with ALK+. None of IHC negative cases were ALK+, demonstrating 100% sensitivity. Eight IHC-positive cases (1 strongly and 7 weakly positive cases) did not show ALK gene rearrangement, resulting in 81.8% specificity. The concordance rate of IHC and FISH is 97.2% (Table 1).

qRT-PCR and VENTANA ALK IHC analysis of discordant cases

VENTANA ALK IHC and qRT-PCR assays were also applied to the remaining 5 of the 12 ALK weakly expressed cases, which were concordant with FISH analysis. These 5 cases were shown to have ALK expression detected by VENTANA ALK IHC, and ALK fusion revealed by qRT-PCR analysis (Table 2).

Clinicopathological characteristics of patients with ALK+