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.

RAF1 rearrangements are common in pancreatic acinar cell carcinomas.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.