Hello, my name is Tim Fielder. I'm a pathology trainee at Royal Prince Alfred Hospital in Sydney and I appreciate the chance to present my poster. I'd like to thank my co-authors, particularly the senior author, Professor Wendy Cooper. I have no conflicts of interest to disclose. A small subset of lung adenocarcinomas are driven by ROS1 rearrangements and can be treated by tyrosine kinase inhibitors. Current practice involves screening for ROS1 IHC before confirmatory molecular testing, but the IHC can often be difficult to interpret with focal positivity and it's difficult to know when to proceed to molecular testing. We looked at all lung adenocarcinomas referred to our department for ROS1 FISH testing over four years. Each case was referred with some ROS1 IHC positivity. We quantitatively scored the IHC in each case with an H-score and compared the H-score to FISH results. An H-score is generated by calculating the staining intensity of tumour cells from 1 to 3 plus and multiplying that by the percentage of positive tumour cells. Of 187 cases, 32 were positive for ROS1 rearrangement by FISH and the remainder were negative. Each and every FISH positive case showed strong and diffuse ROS1 IHC staining, whereas the negative cases were almost all weak and patchy staining. These images show two examples of positive cases. At the top, there's a core biopsy specimen with an H-index of 300 and a cell block specimen at the bottom again with an H-index of 300. So these were by FISH positive for ROS1 rearrangement, as you can see here with split red and green signals or isolated green signals in the majority of tumour cells. This figure shows the spectrum of staining in FISH negative cases. Many of the cases referred to us had only very focal patchy positive staining, whereas some had more diffuse weak to moderate staining. A small subset had diffuse strong and positive staining. It was common for us to see strong staining in reactive pneumocytes, whereas the tumour cells themselves were often weak. This table shows the numerical H-score data. This column is the cases which were rearranged by FISH, and as you can see, the H-score was invariably high, whereas the opposite can be said for the cases that were negative by FISH. If we apply an H-score cut-off of greater than or equal to 200, we capture each and every ROS1 rearranged case for a sensitivity of 100% and 7 of 145 negative cases for a specificity of 95%. Our findings broadly fit in with the previous literature on this subject, although some authors have reported ROS1 rearranged cases with H-scores of less than 200. For example, Cha and colleagues reporting two of 13 cases with a score between 100 and 200, and Yoshida and colleagues, who notably reported a case with an H-index of 5. However, that case had a co-existing EGFR mutation, and it was negative for ROS1 used in transcript on PCR, which cast doubt on the biological relevance of FISH positivity in that case. The other question is why some FISH negative cases have a high H-score. We had seven cases with strong and diffuse ROS1-IHC staining despite negative FISH. It's possible that FISH can generate false negative results with some alterations, for example, small deletions, and ROS1 protein overexpression has also been seen in tumors with other mutations, for example, HER2 alterations. A weakness of our study was the lack of a next-generation sequencing fusion panel to be able to further investigate those cases. The results would have been interesting. In Australia, FISH testing performed on cases with 2-plus intensity receives a government rebate. However, there is some conflict with national consensus guidelines, which suggests FISH testing on cases with any IHC positivity whatsoever. In light of our data, we recommend introduction of an H-score rather than assessment of staining intensity alone when deciding which cases should proceed to FISH. So to summarize, adenocarcinomas with FISH-confirmed ROS1 rearrangements have strong diffuse ROS1-IHC staining. Cases with weak, patchy ROS1-IHC staining are not associated with ROS1 rearrangement by FISH and probably don't require FISH testing. We recommend application of an H-score rather than staining intensity alone when deciding which IHC-positive cases should proceed to molecular analysis. Thank you.

ROS1 IHC

FISH testing

lung adenocarcinomas

ROS1 rearrangements

H-score