Canadian ROS proto-oncogene 1 Study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer,Lung Cancer

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Canadian ROS proto-oncogene 1 Study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer
Lung Cancer ( IF 4.5 ) Pub Date : 2021-08-10 , DOI: 10.1016/j.lungcan.2021.08.003
Carol C Cheung ₁ , Adam C Smith ₁ , Roula Albadine ₂ , Gilbert Bigras ₃ , Anna Bojarski ₄ , Christian Couture ₅ , Jean-Claude Cutz ₆ , Weei-Yuan Huang ₇ , Diana Ionescu ₈ , Doha Itani ₉ , Iyare Izevbaye ₃ , Aly Karsan ₁₀ , Margaret M Kelly ₁₁ , Joan Knoll ₁₂ , Keith Kwan ₁₂ , Michel R Nasr ₁₃ , Gefei Qing ₁₄ , Fariboz Rashid-Kolvear ₁₅ , Harmanjatinder S Sekhon ₁₆ , Alan Spatz ₁₇ , Tracy Stockley ₁ , Danh Tran-Thanh ₂ , Tracy Tucker ₈ , Ranjit Waghray ₁₈ , Hangjun Wang ₁₇ , Zhaolin Xu ₁₉ , Yasushi Yatabe ₂₀ , Emina E Torlakovic ₂₁ , Ming-Sound Tsao ₁

Affiliation

Laboratory Medicine Program, University Health Network, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
Department of Pathology, Centre hospitalier de l'Université de Montréal, Montréal, QC, Canada.
Laboratory Medicine Department, University of Alberta, Edmonton, AB, Canada.
Department of Pathology and Laboratory Medicine, Health Sciences North, Sudbury, ON, Canada.
Institut universitaire de cardiologie et de pneumologie de Québec-Université Laval, Quebec City, QC, Canada.
Department of Pathology and Molecular Medicine, McMaster University Health Sciences Centre and McMaster University, Hamilton, ON, Canada.
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada; Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Science Center, ON, Canada.
Department of Pathology and Laboratory Medicine, BC Cancer, Vancouver, BC, Canada.
Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; Department of Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, USA.
Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada.
Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada.
Department of Pathology and Laboratory Medicine, London Health Sciences Centre, London, Ontario, Canada.
Department of Pathology, Shared Health Manitoba, University of Manitoba, Winnipeg, MB, Canada; Department of Pathology SUNY Upstate Medical University, Syracuse, NY, USA.
Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary, AB, Canada, and Calgary Laboratory Services, Calgary, AB, Canada.
Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary, AB, Canada, and Calgary Laboratory Services, Calgary, AB, Canada; Department of Pathology and Laboratory Medicine, Johns Hopkins Medicine, Johns Hopkins All Children's Hospital, Baltimore, MD, USA.
Department of Pathology and Laboratory Medicine, The Ottawa Hospital and ORLA, University of Ottawa, Ottawa, ON, Canada.
Divisions of Pathology and Molecular Genetics, McGill University Health Center and McGill University, Montreal, QC, Canada.
Department of Pathology and Laboratory Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Dept. of Pathology, Queen Elizabeth II Health Sciences Centre and Dalhousie University, Halifax, NS, Canada.
Department of Diagnostic Pathology, National Cancer Center, Tokyo, Japan.
Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan and Saskatchewan Health Authority, Saskatoon, SK, Canada.

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 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 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 rearrangement 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.

中文翻译：

加拿大 ROS 原癌基因 1 研究 (CROS) 在非小细胞肺癌中多机构实施 ROS1 检测

携带ROS原癌基因 1 ( ROS1 ) 基因重排的非小细胞肺癌 (NSCLC) 患者对酪氨酸激酶抑制剂 (TKI)克唑替尼表现出显着的反应。当前的最佳实践指南建议对所有晚期非鳞状 NSCLC 患者也进行ROS1基因重排检测。几项研究表明，使用 D4D6 抗体的 ROS1 免疫组织化学 (IHC) 可用于筛查 ROS1 融合阳性肺癌，检测结果显示高灵敏度但中等至高特异性。然后使用分离荧光原位杂交 (FISH) 测试来确认 ROS1 基因重排的存在。加拿大ROS1 (CROS) 研究的目标是协调ROS1实验室开发了测试 (LDT)，通过使用 IHC 和 FISH 检测来检测加拿大病理实验室的ROS1重排肺癌。

表达不同水平ROS1（高、低、无）的细胞系用于校准 IHC 方案，之后参与实验室在 24 个 NSCLC 病例的参考集（9 个ROS1重排肿瘤和 15 个ROS1非重排肿瘤）上运行校准方案通过鱼）。使用集中读数比较结果。就染色的细胞定位、染色强度、非肿瘤细胞中染色的存在、非特异性染色（例如坏死、细胞外物质、其他）的存在和阳性细胞百分比评估染色的载玻片。还确定了每个肿瘤的 H 分数。

通过使用检测下限 (LOD) 作为任何阳性 U118 细胞系或 HCC78 细胞系的 H 分数为 200，实现了分析灵敏度和特异性的协调。ROS1 IHC 测试（相对于 FISH）使用参考病例调整后的 H 分数读数分别实现了高达 100% 和 99% 的总体诊断灵敏度和特异性。

该研究证实，LDT ROS1 IHC 检测对于检测NSCLC 中的ROS1重排具有高度敏感性。由于 NSCLC 可以在 FISH 阴性病例中证明ROS1 IHC 阳性，因此 IHC 检测的特异性程度，尤其是在高度敏感的方案中，主要取决于读数截止阈值。由于ROS1 IHC 是 NSCLC 中罕见重排的筛选分析，我们建议调整读数阈值以平衡特异性，而不是降低方案的整体分析和诊断灵敏度。

更新日期：2021-08-10

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