Testing for ROS1 in non-small cell lung cancer: a review with recommendations

The screening strategy for ROS1 rearrangement was developed based on the experience of ALK testing in lung adenocarcinomas [17, 23, 32]. While ROS1 alterations may be detected with a variety of techniques, most laboratories rely on FISH assays using a dual-colour break-apart probe design. These involve labelling the 3′ (centromeric) part of the fusion breakpoint with one fluorochrome and the 5′ (telomeric) part with another fluorochrome. It is important to choose a 3′ probe colour [16, 22, 35‐37] that allows ROS1 (most commonly a green 3′ fluorochrome) and ALK (an orange 3′ fluorochrome) tests to be distinguished [38], particularly if the two tests are to be run together on one slide, in parallel.

There are two positive ROS1 rearrangement patterns. One is the break-apart pattern (‘classic’ pattern) with one fusion signal (native ROS1) and two separated 3′ and 5′ signals. The other positive pattern is an isolated 3′ signal pattern, usually an isolated green signal, (‘atypical’ pattern) with one fusion signal (native ROS1) and one 3′ signal without the corresponding 5′ signal (Fig. 2). Table 3 summarises the criteria for ROS1 FISH interpretation in NSCLC [23, 32, 35, 36].

For optimal FISH results, there are a number of relevant factors. Use of sections older than 6 months may result in poor hybridisation. In the post-analytic phase, it is important that only intact tumour cells with non-overlapping nuclei are scored. Furthermore, the use of an automated software system (e.g. BioView Duet system, Rehovot, Israel) can facilitate FISH scoring. It should be noted that FISH testing for ROS1 (and ALK) is not restricted to histological tissue sections, but is also applicable to cytological specimens [39, 40].

Given the rarity of ROS1 rearrangements in NSCLC, screening of tumours by IHC may allow unnecessary FISH analysis in ROS1-negative cases to be avoided and thus dramatically reduce the cost of testing. Based on available data, IHC is an effective screening tool to detect ROS1-positive NSCLC, with a sensitivity of 100 % in most studies and a variable specificity ranging from 92 to 100 %, depending on the threshold used to define positivity [20, 36, 37, 41‐45]. These results are based on the use of the ROS1 (D4D6) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) applied at dilutions ranging from 1:50 to 1:1000 with various antigen retrieval methods and use of different amplification and detection systems, in automated instruments or manually. Tumour specimens with known ROS1 rearrangement, or a cellblock of the HCC78 cell line harbouring the SLC34A2-ROS1 fusion gene, can serve as positive controls [20] (Figs. 3a and 4a). In contrast to ALK, where the ganglion cells of the appendix serve as an adequate external control, there is currently no good external benign tissue control for ROS1.

Currently, there is no universally accepted system for how to score IHC results. The thresholds used include either any staining above faint background (if present) or moderate or strong staining (2+/3+). Another option is the use of an H-score with optimal threshold for ROS1 positivity defined as >100 [40] or >150 [43]. Notably, weak and focal staining was found in 31 % of 253 ROS1 wild-type lung carcinomas in one study [44]. However, this had hardly any influence on specificity when appropriate thresholds of positivity were used (i.e. H-score >150). Thus, none of the reported scoring methods have been shown to be clearly superior to the others since all resulted in very good to excellent correlation with FISH results.

Positive ROS1 IHC typically reveals finely granular cytoplasmic staining (Fig. 3). However, the staining pattern may depend on the function and subcellular location of the gene fusion partner [44, 46]. Globular ROS1 immunoreactivity has been described in tumour specimens with the CD74-ROS1 fusion, and membranous staining has been observed in tumours with the EZR-ROS1 fusion [41, 44]. Interestingly, ROS1 expression levels in ROS1-positive lung cancers and cell lines can vary from cell to cell, suggesting dynamic ROS1 protein expression despite homogeneous presence of ROS1 gene rearrangement (Figs. 3 and 4). Detection of ROS1 protein expression in ROS1-positive adenocarcinomas with signet ring cells is challenging since the cytoplasm is largely replaced by non-reactive mucin [44]. The same pitfall has already been shown for ALK IHC [47]. One should be aware that weak ROS1 expression is occasionally detectable in non-neoplastic hyperplastic type II pneumocytes (Fig. 3e, h) and in alveolar macrophages. In bone metastases, there is strong granular cytoplasmic staining of osteoclast-type giant cells (Fig. 3f).

Lung cancer is often diagnosed by cytology alone, necessitating ROS1 testing in cytological specimens. For cellblocks, the same IHC protocols can be used as for formalin-fixed paraffin-embedded (FFPE) tissue specimens. In the laboratory of one of the authors (L.B.), ROS1 IHC on the BondMax immunostainer is routinely used on conventional, ethanol-fixed and Papanicolaou-stained cytological specimens, including smears or cytospin preparations (Fig. 4). Notably, in the case of limited cytological material, FISH for confirmation of a positive IHC result can be applied to the immunostained slide if 3-amino-9-ethylcarbazole (AEC) was used as a red chromogen. Although IHC on cytological specimens is common practice in many laboratories, immunocytochemistry performed on smears and/or cytospin slides may be much more influenced by various pre-analytical factors [48, 50]. Thus, it should be performed only in laboratories with experience and appropriate quality assurance in place. The simultaneous use of cell blocks would allow testing sequentially for several biomarkers.

ROS1 IHC has a great advantage over FISH in that it can detect rare positive cells or cell groups within a majority of non-neoplastic reactive cells that would be easily missed by FISH. This can be particularly helpful in cytological specimens where architectural tissue context is lost. Although it has been proposed to score ROS IHC only in specimens containing ≥20 tumour cells [41], a positive result in even only a few clearly neoplastic cells can be considered diagnostic.

In addition to FISH and IHC, a number of non-in situ approaches based on real-time PCR (RT-PCR) or NGS have been developed for the detection of ROS1 gene rearrangements. RT-PCR assays require multiple specific primer sets to discriminate amongst known fusion variants, which can be confirmed by subsequent sequencing [50]. The breakpoints of ROS1 are located at exons 32, 34, 35 and 36, and the most frequent ROS1 fusion partners include SLC34A2, CD74, TPM3, SDC4, EZR, LRIG3, FIG or GOPC, MSN, KDELR2 and CCDC6 [18, 19, 21, 22, 51]. RT-PCR has been successfully utilised to identify positive cases with a sensitivity of 100 % and a specificity of 85–100 %, using FISH as the reference standard method [37, 42]. Multiplex RT-PCR is easy to perform, rapid and relatively inexpensive but may be challenging using RNA extracted from FFPE samples [52]. In addition, as the list of ROS1 fusion partners is quite large and still growing, RT-PCR is likely to miss rare variants. These reasons have limited the use of the technique in clinical practice. Recently, a very sensitive RT-PCR-based method was devised to detect the overexpression of 3′ regions of fusion transcripts involving tumour genes constitutionally repressed or expressed at very low levels [53]; this approach has been successfully applied to ALK gene fusions in lung cancer [53, 54]. Unfortunately, this method cannot be easily applied to ROS1, since the gene is also expressed in normal and hyperplastic lung tissue [15, 55]. An alternative transcript-based method for detecting ROS1 fusion genes is also available. The NanoString assay, capable of detecting known fusion gene transcripts and employing a dual capture and reporter probe system, provides a convenient and commercially available assay that has shown good concordance with FISH and IHC results for ROS1 [50, 55].

A series of innovative approaches to detect gene fusions in multiple targets has been developed using NGS (Table 4). It is remarkable that some of these comprehensive assays require as little as 10 ng of RNA [56], with relatively low failure rates in paraffin-embedded tissue (5.6 % in the authors’ experience [unpublished data]). A very sensitive NGS technique to assess ROS1 and other gene rearrangements in lung cancer is anchored multiplex PCR that targets only the gene of interest, allowing the detection of the specific alteration irrespective of fusion partner. Validation of a gene rearrangement panel using 319 FFPE samples showed 100 % sensitivity and 100 % specificity compared with reference assays [51].

These promising results suggest potential application of non-in situ methodologies in clinical practice, as stand-alone methods or as complementary tests within algorithms for the selection of patients to be treated with ROS1, RET or NTRK inhibitors [57]. However, published data for these assays are still limited.

There is good correlation between FISH and IHC using clone D4D6 with a highly sensitive amplification kit. Although some discrepant cases have been reported, ROS1 testing by IHC seems to be highly sensitive, but less specific, also when compared with ALK IHC for detection of the corresponding gene rearrangement. As suggested by others [41], IHC testing of specimens containing at least 20 tumour cells and application of an H-score cut-off of >100 are highly concordant with ROS1 rearrangement by FISH or RT-PCR.

Currently, there is very limited published information on the concordance of IHC, in situ hybridisation (ISH) and non-in situ tests for the detection of ROS1 gene rearrangements in lung adenocarcinoma [36, 37, 42, 58]; less than 30 cases with gene rearrangements have been subjected to comparative study of the three methods. Four of the ROS1 tests currently hold in vitro diagnostic (IVD) and CE-marked status (Table 5). The general consensus seems to be that IHC, ISH and non-in situ methods all are promising for the detection of ROS1-positive cases, with concordance rates well above 90 %. Preliminary conclusions from limited studies suggest a role for IHC as a screening tool, but so far the lack of an IVD-classified IHC assay is problematic. Advantages of RT-PCR analysis include the highest sensitivity reported and the ability to identify translocation partners. On the other hand, there are potential issues around the quality and quantity of mRNA that may be obtained from routine, FFPE NSCLC diagnostic tissues. Furthermore, familiarity and availability of FISH as a technique in detecting other markers, such as ALK, is also important. Perhaps the use of more than one technique could be of value until further experience of testing and companion diagnostics with IVD status have emerged.

With ISH techniques, the case for study serves as a control when consistently presenting signals, both in tumour cells and in the accompanying normal cells (lymphocytes, fibroblasts, non-neoplastic lung epithelium). A pre-hybridisation assessment of digestion is useful in difficult samples (e.g. very small biopsies with low tumour content).

With IHC, ROS1 protein expression may also be detected in normal cells, namely histiocytes/giant cells, reactive type II pneumocyte hyperplasia and bronchiolar metaplasia at the tumour periphery or in subpleural areas. In most cases, the expression in these cells is weak to moderate (1+/2+ in intensity), and it is unclear whether protein stability may be affected by pre-analytical variables (e.g. time of fixation) [43]. To control for appropriate analytical conditions of ROS1 testing by IHC, it is mandatory to include a piece of tissue from a ROS1 FISH-positive tumour on the same slide of the neoplasm of interest or on a separate slide to use in the same run.

Regarding the ROS1 epitope, the influence of pre-analytical factors has not been investigated systematically, but experiences from our group and several others [36, 61] show that the protein is relatively stable and may be detected reliably by IHC. In addition, the corresponding genomic alterations can be reproducibly detected by ISH [62]. To improve nucleic acid stability, new techniques of fixation may become useful [63]. Nonetheless, attention should be paid to a number of basic requirements in order to avoid false-negative results.

For the successful treatment of patients, it is of great importance that molecular test results are accurate, highly reliable, clearly understandable to the clinician and reported within an acceptable turnaround time [59]. In 2012, the European Society of Pathology (ESP) proposed an external quality assessment (EQA) scheme to promote high-quality biomarker testing in NSCLC for EGFR mutation analysis and ALK rearrangement detection. From 2014 onwards, ROS1 testing was also included [65]. The EQA was performed at the beginning of the development of ROS1 testing. The rate of false negativity for IHC on a limited number of ROS1-positive cases was approximately 15 %. The rate of false positivity was <10 %. For FISH, although the number of evaluable cases was limited, no false-negative scores were present in a ROS1-positive control cell line. Overall, at an early stage of ROS1 testing, the EQA showed promising results, emphasising the need for regular EQA monitoring.