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ROS1 Immunohistochemistry (IHC)

ROS1 IHC preanalytical considerations

  • Place specimen in fixative as quickly as possible to minimise antigen degradation as a result of cold ischaemia
  • Fixation in 10% neutral buffered formalin for at least 6 hours and for as long as 48–72 hours.
  • Store tissue embedded in paraffin in a climate-controlled environment (25°C in dry conditions)
  • Once a 4- to 6-μm tissue section is cut onto glass slides, exposure to the air may lead to oxidative damage and signal degradation
  • Antigenicity of cut tissue sections likely to decline after 3 months

IASLC Atlas of ALK and ROS1 Testing in Lung Cancer, IASLC, Colorado, USA
Dietel M et al. Thorax. 2016;71:177-84.

ROS1 IHC protocols using D4D6® rabbit monoclonal antibody (Cell Signalling Technology)

  • ROS1 IHC protocol for antibody clone D4D6 for Papanicolaou-stained cytology smears or cytospins

    LEICA BOND-MAX: BOND Polymer Refine
    Detection Kit with AEC

    Cell Conditioning:
    HIER
    ER2: 10 minutes (Epitope Retrieval
    solution 2) 80°C
    Ab incubation: 30 minutes 20-25°C
    Amplification:
    Included in kit
    Post Primary 15 minutes 20-25°C
    Polymer 15 minutes 20-25°C
    Counterstain [Hematoxylin] 5 minutes 20-25°C

     LEICA BOND-MAX: BOND Polymer Refine Detection Kit with
    AEC for Cytology smears

    1. Staining: AEC5
    2. No Deparaffinization for Cytology
    3. Cell Conditioning: HIER: ER2
    4. EPITOPE RETRIEVAL 2: 10 minutes 80°C
    5. PEROXIDE BLOCK: 5 minutes 20-25°C
    6. PRIMARY ANTIBODY ROS1 CELL SIGNALING (clone D4D6) 1/200
    7. Apply primary antibody and incubate for 30 minutes 20-25°C
    8. POST PRIMARY incubate for 15 minutes 20-25°C
    9. POLYMER incubate for 15 minutes 20-25°C
    10. CHROMOGEN AEC incubate for 10 minutes 20-25°C
    11. Counterstain HEMATOXYLIN
    12. Apply one drop of HEMATOXYLIN [in Kit included] and incubate for 5 minutes 20-25°C
  • BenchMark Ultra protocol or histological specimens

    PROTOCOL SUMMARY
    Procedure: U OptiView DAB IHC v5 (v1.00.0117)

    BenchMark ULTRA IHC/ISH Staining Module
    Provided by Prof. Lukas Bubendorf, Pathologie USB Basel, Switzerland


    1. Paraffin [Selected]
    2. Deparaffinization [Selected]
    3. Warmup Slide to [72°C] from Medium Temperatures (Deparaffinization)
    4. Cell Conditioning [Selected]
    5. Ultra CC1 [Selected]
    6. Warmup Slide to [100°C], and Incubate for 4 minutes (Cell Conditioner #1)
    7. CC1 8 Min [Selected]
    8. CC1 16 Min [Selected]
    9. CC1 24 Min [Selected]
    10. CCI 32 Min [Selected]
    11. CC1 40 Min [Selected]
    12. CC1 48 Min [Selected]
    13. Pre Primary Peroxidase Inhibit. [Selected]
    14. Primary Antibody [Selected]
    15. Apply Coverslip, One Drop of [PREP KIT 6] (Antibody), and Incubate for [0 Hr 32 Min]
    16. Counterstain [Selected]
    17. Apply One Drop of [HEMATOXYLIN] (Counterstain), Apply Coverslip, and Incubate for [8 Minutes]
    18. Post Counterstain [Selected]
    19. Apply One Drop of [BLUING REAGENT] (Post Counterstain), Apply Coverslip, and Incubate for [4 Minutes]
  • BenchMark XT protocol on histological specimens

    PROTOCOL SUMMARY
    Procedure: XT OptiView DAB IHC v4

    BenchMark XT IHC/ISH Staining Module
    Provided by Prof. Lukas Bubendorf, Pathologie USB Basel, Switzerland


    1. Paraffin [Selected]
    2. Deparaffinization [Selected]
    3. Cell Conditioning [Selected]
    4. CC1 [Selected]
    5. CC1 8 Min [Selected]
    6. CC1 16 Min [Selected]
    7. CC1 24 Min [Selected]
    8. CC1 32 Min [Selected]
    9. CC1 40 Min [Selected]
    10. Pre Primary Peroxidase Inhibit. [Selected]
    11. Primary Antibody [Selected]
    12. Apply Coverslip, One Drop of [PREP KIT 6] (Antibody), and Incubate for [0 Hr 24 Min]
    13. OptiView Amplification [Selected]
    14. Apply One Drop of OV AMP H202 and One Drop of OV AMPLIFIER, Apply Coverslip, Incubate for [4 Minutes]
    15. Apply One Drop of OV AMP MULTIMER, Apply Coverslip, and Incubate for [4 Minutes]
    16. Counterstain [Selected]
    17. Apply One Drop of [HEXATOXYLIN] (Counterstain), Apply Coverslip, and Incubate for [8 Minutes]
    18. Post Countertain [Selected]
    19. Apply One Drop of [BLUING REAGENT] (Post Counterstain), Apply Coverslip, and Incubate for [4 minutes]

    Protocols provided by Prof. Lukas Bubendorf, Pathologie USB Basel, Switzerland

OTHER PUBLISHED PROTOCOLS

Author

N total

N ROS1+

Sensitivity

Specificity

Cut-off

Sholl LM, et al. Am J Surg Pathol 2013;7:1441-9

218

12

100%

92%

2+ & 3+

Yoshida A, et al. Mod Pathol 2014;27:711-20;

270

17

(100%)

94%

(69%)

98%

(69%)

H-score ≥150

(any positivity)

Cha YJ, et al. PLoS One 2014;9;

330

13

100%

95–97.8%

2+ & 3+, or

H-score ≥100

Wu J, et al. BMC Cancer 2016;16:599

238

10

100%

100%

H-score ≥150

 

Selinger CI, et al. Histopathol 2017;70:402–411

382

108

100%

88%*

2+ & 3+

D4D6 rabbit monoclonal antibody, Cell Signaling

*76% in retrospective cohort (100% if cut-off 3+)

Positive controls for ROS1 IHC

  • To control for appropriate analytical conditions, it is mandatory to include a tumour specimen with known (FISH-positive) ROS1 rearrangement on the same slide of the neoplasm of interest or on a separate slide in the same run
  • 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

Cell lines:

  • A paraffin-embedded cell block of the ROS1-rearranged cell line HCC-78 harboring the SLC34A2-ROS1 fusion gene can also be used, however, use of a positive control that expresses the target at a low level will help to confirm sensitivity
  • The glioblastoma cell line U-118 MG, which contains a FIG (GOPC)-ROS1 fusion expresses ROS1 at a low level by Western blot and IHC

IASLC Atlas of ALK and ROS1 Testing in Lung Cancer, IASLC, Colorado, USA

Bubendorf L et al. Virchows Arch. 2016;469:489–503.

Rimkunas VM et al, Clin Cancer Res 2012;18:4449–57.