Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib

Lung cancer is one of the most common malignant tumors worldwide [1]. Detecting driver genes in patients with non-small cell lung cancer (NSCLC) is the new standard for clinical decision-making [2, 3]. Detecting somatic mutations within epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) driver gene status has become a diagnostic routine for lung adenocarcinoma [4‐6]. Therefore, detecting driver gene mutation status is the key to guide therapeutic decisions in clinical practice.

Crizotinib, a dual ALK–MET inhibitor, was approved by the Food and Drug Administration (FDA) in the United States for the treatment of advanced-stage NSCLC harboring an ALK rearrangement [7, 8]. One key issue in the detection of altered ALK is the method that best defines ALK status in a clinical setting. Currently, the diagnostic gold standard for detecting ALK rearrangements is break-apart fluorescence in situ hybridization (FISH), which is able to detect a large majority of ALK rearrangements, especially EML4-ALK, and has been clinically validated in crizotinib clinical trials [9‐11]. ALK FISH, however, is fraught with technical and financial problems, including FISH signal instability and scoring difficulties. An alternative method for determining ALK rearrangements in NSCLC is a reverse transcription-polymerase chain reaction (RT-PCR). The Chinese FDA has approved the ADx EML4-ALK fusion diagnostic kit to be used for RT-PCR to detect ALK rearrangements; this assay has also been accepted by the Chinese Anti-cancer Association, but the requirement for fresh frozen tissue samples for extracting RNA has limited the application in clinical practice. Thus, the immunohistochemical method is a standard method to identify ALK protein overexpression in NSCLC. VENTANA immunohistochemistry (IHC) is relatively inexpensive, quick, and is performed routinely in most surgical pathology practices. VENTANA IHC was approved to detect ALK protein in pathology practice in European and some Asian countries. Currently, the response to crizotinib in NSCLC patients overexpressing ALK detected by VENTANA IHC is largely unknown. Recent developments in next-generation sequencing (NGS) of DNA and RNA have created a new opportunity for simultaneous detection of a large number of gene fusions with known and unknown partner genes and parallel detection of gene mutations [12‐15]. The results of successful screening for oncogenic fusions using the NGS method have been recently reported [13].

To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of screening for ALK rearrangements in NSCLC. Seventeen inconsistent cases with VENTANA IHC and RT-PCR underwent targeted NGS. Therefore, we investigated the response to crizotinib among Chinese NSCLC patients who overexpressed ALK as detected by VENTANA IHC, and compared the clinical value with the RT-PCR method.

ALK rearrangements in patients with NSCLC are highly sensitive to crizotinib treatment [18]. As first-line treatment, crizotinib has an ORR of 74% and the median PFS is 10.9 months [6]. Therefore, identification of appropriate patients with reliable detection methods is important for targeted therapy. Previous studies have demonstrated that VENTANA IHC is a highly sensitive and specific assay for detection of ALK gene status, and is a feasible alternative to the ALK FISH assay [19‐21]. RT-PCR is an alternative method that is rapid and convenient to perform [22]; however, the requirement of fresh frozen tissue samples for extracting RNA by the RT-PCR assay has limited its application in clinical practice. In the current study, we compared VENTANA IHC with the RT-PCR assay to detect ALK rearrangements and report for the first time the response and survival of crizotinib for Chinese patients with EML4-ALK positive advanced NSCLC detected by VENTANA IHC and RT-PCR. In the enrolled 1720 patients, we identified 10.27% (172/1674) patients had ALK positive by VENTANA IHC method, 12.73% (41/322) patients had ALK rearrangements by RT-PCR method, and 9.42% (26/276) patients were both positive by VENTANA IHC and RT-PCR. It has been reported that ALK rearrangements range from 2 to 7% among unselected Caucasian NSCLC patients [23, 24]. The frequency has been reported to be as high as 5–10% and is higher in the Asian population [25‐27]. The frequency of detection by RT-PCR in the current study was higher than VENTNANA IHC. Rosell et al. [28] showed that RT-PCR detected more cases with the EML4-ALK fusion gene (12.5%) than IHC (6.7%) among 200 NSCLC patients, and based on routine examination by the two techniques, the EML4-ALK rearrangements can be detected more frequently by RT-PCR. The VENTANA IHC assay is performed routinely in most surgical pathology practices and IHC has been demonstrated as a reliable pre-screening test for detecting lung cancer in clinical practice. In addition, we observed an ORR of 59.09%, DCR of 80.30%, and median PFS of 9.0 months in 66 ALK-positive patients. The ORR was 65.90% and the DCR was 86.36% in the 44 patients in whom an ALK translocation was confirmed by VETNANA IHC. The ORR was 55.88% and the DCR was 76.47% by RT-PCR in 34 ALK-positive patients. The median PFS of VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively (p = 0.630). Although our study included first-, second-, and later-line crizotinib therapy, the median PFS was within the range of 7.7–10.9 months, as reported in relevant clinical trials [6, 10, 29]. Interestingly, we also observed responses to crizotinib in two patients who had positive ALK fusion by VENTANA IHC, but not by RT-PCR. Another case which was ALK-positive by RT-PCR, but negative by VENTANA IHC, did not show a good response to crizotinib treatment. Therefore, VENTANA IHC is a rapid and relatively inexpensive method for diagnosing ALK-rearranged NSCLC. RT-PCR may be highly sensitive, but the specificity of RT-PCR as a screening tool is likely to be extremely high and low abundance results accompanied other genes may occur with this highly sensitive technique [28]. The application of VENTANA IHC is more moderate than RT-PCR as a screening method to detect ALK status in clinical practice and patients could benefit from crizotinib more so than RT-PCR.

In the current study, there were six patients with ALK VENTANA IHC-positive and RT-PCR-negative, demonstrating ALK rearrangement revealed by NGS. Another 6 patients who had primary resistance to crizotinib therapy were also analyzed by NGS. Our results suggest that 4 patients were identified to have EML4-ALK variants, including E6; A20, E13; A20, E14; A20, and E18; A20. Two patients were identified to have non-EML4-ALK fusions; one patient with a KCL1-ALK fusion, which had been previously reported in the literature [30], and one patient with a FBXO36-ALK fusion who received crizotinib therapy with a PFS of 21.2 months and an OS of > 46.7 months. NGS revealed a new ALK partner gene, FBXO36, which is the first report in NSCLC worldwide, and it has good response to crizotinib. Currently, developments in NGS have created a new method for the simultaneous detection of a large number of gene fusions with known and unknown genes and gene mutations [12, 14, 15]. Pekar-Zlotin et al. [12] reported a 42.9% sensitivity and 97.7% specificity for ALK FISH when compared to NGS DNA-based platform for the detection of ALK gene rearrangements. Dacic et al. [31] also demonstrated significant concordance between IHC and NGS in cases discordant between NGS and FISH. VENTANA IHC detects ALK expression for ALK fusion genes independent of variant and fusion partners. Therefore, the VENTANA IHC method is highly recommended in routine pathologic diagnosis.

Although our results are significant, we recognize that there are limitations to the study. First, a major limitation was the retrospective design. Second, because of insufficient samples or DNA, we did not assess all tissues by VENTANA IHC and RT-PCR.

Our study suggests that VENTANA IHC may be a reliable and initial screening approach for assessment of ALK rearrangements in patients with NSCLC. In addition to RT-PCR, VENTNANA IHC also identified patients who responded to crizotinib and are RT-PCR negative. NGS as a technique for detecting ALK gene fusions and primary resistance to ALK TKI warrant further study.