Canadian ROS proto-oncogene 1 study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Cheung CC ，Smith AC ，Albadine R ，Bigras G ，Bojarski A ，Couture C ，Cutz JC ，Huang WY ，Ionescu D ，Itani D ，Izevbaye I ，Karsan A ，Kelly MM ，Knoll J ，Kwan K ，Nasr MR ，Qing G ，Rashid-Kolvear F ，Sekhon HS ，Spatz A ，Stockley T ，Tran-Thanh D ，Tucker T ，Waghray R ，Wang H ，Xu Z ，Yatabe Y ，Torlakovic EE ，Tsao MS ... -  《-》

On the relevance of a testing algorithm for the detection of ROS1-rearranged lung adenocarcinomas.

ROS1 proto-oncogene translocations define a new molecular subgroup in non-small cell lung cancers (NSCLC) and are associated with a response to the MET/ALK inhibitor, crizotinib. These rearrangements are described in 0.9-1.7% NSCLC, in wild-type EGFR, KRAS and ALK ("triple negative") lung adenocarcinomas. Rapid and efficient identification of these alterations is thus becoming increasingly important. In this study, 121 triple negative lung adenocarcinomas were screened by both IHC with the ROS1 D4D6 antibody, and FISH using two commercially available ROS1 break-apart probes. To address a possible cross-reactivity of the ROS1 antibody with other protein kinase receptors, we screened 80 additional cases with known EGFR, KRAS, PI3KCA, BRAF, HER2 mutations or ALK-rearrangement. We diagnosed 9 ROS1-rearranged adenocarcinomas, with both a positive FISH result (51-87% rearranged nuclei) and a positive IHC staining (2+/3+ cytoplasmic staining). Only one of the ROS1-positive FISH cases was characterized by a classical split pattern, the others showed a variant pattern, most commonly involving a loss of the 5' telomeric probe. Considering a positivity threshold of 2+ stained cells, the sensitivity of the ROS1 D4D6 antibody compared to FISH was 100% and the specificity 96.9%, as two HER2-mutated tumors were positive with D4D6 antibody, without any translocation in FISH. All the ROS1-positive cases were at an advanced stage, arising in never or light smokers. They were mainly solid cribriform and acinar adenocarcinomas, with signet ring cells noted in 5 cases, and calcifications in 3 cases. One positive case was an invasive mucinous carcinoma. Our results show that a screening algorithm based on an IHC detection of ROS1 fusion proteins, confirmed if positive or doubtful by a ROS1 break-apart FISH assay, is pertinent in advanced "triple negative" lung adenocarcinomas, since the prevalence of ROS1-positive cases in this selected population reaches 7.4% in our series.

Mescam-Mancini L ，Lantuéjoul S ，Moro-Sibilot D ，Rouquette I ，Souquet PJ ，Audigier-Valette C ，Sabourin JC ，Decroisette C ，Sakhri L ，Brambilla E ，McLeer-Florin A ... -  《-》

ROS1 rearrangements in non-small cell lung cancer: screening by immunohistochemistry using proportion of cells staining without intensity and excluding cases with MAPK pathway drivers improves test performance.

Therapeutically actionable ROS1 rearrangements have been described in 1-3% of non-small cell lung cancer (NSCLC). Screening for ROS1 rearrangements is recommended to be by immunohistochemistry (IHC), followed by confirmation with fluorescence in situ hybridisation (FISH) or sequencing. However, in practise ROS1 IHC presents difficulties due to conflicting scoring systems, multiple clones and expression in tumours that are wild-type for ROS1. We assessed ROS1 IHC in 285 consecutive cases of NSCLC with non-squamous histology over a nearly 2-year period. IHC was scored with ROS1 clone D4D6 (n=270), clone SP384 (n=275) or both clones (n=260). Results were correlated with ROS1 break-apart FISH (n=67), ALK status (n=194), and sequence data of EGFR (n=178) and other drivers, where possible. ROS1 expression was detected in 161/285 cases (56.5%), including 13/14 ROS1 FISH-positive cases. There was no ROS1 expression in one ROS1 FISH-positive case in which sequencing detected an ALK-EML4 fusion, but not a ROS1 fusion. The other 13 ROS1 FISH-positive cases showed moderate to strong staining with both IHC clones. However, one case with a TPM3-ROS1 fusion would have been scored as negative with SP384 and D4D6 clones by some previous criteria. ROS1 expression was also detected in 58/285 cases (20.4%) that had driver mutations in genes other than ROS1. A sensitivity of 100% for detecting a ROS1 rearrangement by FISH was achieved by omitting intensity from the IHC scoring criteria and expression in >0% cells with D4D6 or in ≥50% cells with SP384. Excluding cases with driver events in any MAPK pathway gene (e.g., in ALK, EGFR, KRAS, BRAF, ERBB2 and MET) substantially reduced the number of cases proceeding to ROS1 FISH. Only 15.9% of MAPK-negative NSCLC would proceed to FISH for an IHC threshold of >0% cells with D4D6, with a specificity of 42.4%. For a threshold of ≥50% cells with SP384, only 18.5% of MAPK-negative cases would proceed to FISH, with a specificity of 31.4%. Based on our data we suggest an algorithm for screening for ROS1 rearrangements in NSCLC in which ROS1 FISH is only performed in cases that have been demonstrated to lack activating mutations in any MAPK pathway gene by comprehensive sequencing and ALK IHC, and show staining at any intensity in ≥50% of cells with clone SP384, or >0% cells with D4D6.

Prall OWJ ，Browning J ，Nastevski V ，Caporarello S ，Bates B ，Hewitt CA ，Arenas A ，Lamb G ，Howlett K ，Arnolda R ，Adeloju R ，Stuart S ，Xu H ，Fellowes A ，Fox SB ... -  《-》

Comparison of detection methods and follow-up study on the tyrosine kinase inhibitors therapy in non-small cell lung cancer patients with ROS1 fusion rearrangement.

Wu J ，Lin Y ，He X ，Yang H ，He P ，Fu X ，Li G ，Gu X ... -  《BMC CANCER》

Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohistochemistry with FISH confirmation is an effective method to identify this rare target.

To assess the prevalence of ROS1 rearrangements in a retrospective and prospective diagnostic Australian cohort and evaluate the effectiveness of immunohistochemical screening. A retrospective cohort of 278 early stage lung adenocarcinomas and an additional 104 prospective non-small-cell lung cancer (NSCLC) cases referred for routine molecular testing were evaluated. ROS1 immunohistochemistry (IHC) was performed (D4D6 clone, Cell Signaling Technology) on all cases as well as fluorescence in-situ hybridization (FISH) using the ZytoVision and Abbott Molecular ROS1 FISH probes, with ≥15% of cells with split signals considered positive for rearrangement. Eighty-eight cases (32%) from the retrospective cohort showed staining by ROS1 IHC, and one case (0.4%) showed ROS1 rearrangement by FISH. Nineteen of the prospective diagnostic cases showed ROS1 IHC staining, 12 (12%) cases of which were confirmed as ROS1 rearranged by FISH. There were no ROS1 rearranged cases that showed no expression of ROS1 with IHC. The ROS1 rearranged cases in the prospective cohort were all EGFR wild-type and anaplastic lymphoma kinase (ALK) rearrangement-negative. The sensitivity of ROS1 IHC in the retrospective cohort was 100% and specificity was 76%. ROS1 rearrangements are rare events in lung adenocarcinomas. Selection of cases for ROS1 FISH testing, by excluding EGFR/ALK-positive cases and use of IHC to screen for potentially positive cases, can be used to enrich for the likelihood of identifying a ROS1 rearranged lung cancer and prevent the need to undertake expensive and time-consuming FISH testing in all cases.

Selinger CI ，Li BT ，Pavlakis N ，Links M ，Gill AJ ，Lee A ，Clarke S ，Tran TN ，Lum T ，Yip PY ，Horvath L ，Yu B ，Kohonen-Corish MR ，O'Toole SA ，Cooper WA ... -  《-》