Multiplex IHC Analysis of Tumor Biopsies

Tissue biopsies were obtained at baseline and at the end of the third cycle of therapy (after 2 doses of CY/nivolumab/GVAX and 1 dose of nivolumab/CRS-207 in Arm A and 2 doses of CY/GVAX and 1 dose of CRS-207 in Arm B). A blinded study pathologist selected biopsies containing >30% tumor cellularity for multiplex immunohistochemistry (IHC). Chromogenic sequential IHC was conducted on 5-micron formalin-fixed paraffin-embedded tissue sections as previously described (13 (link)). Three 12-plex multiplex IHC panels containing markers of lymphoid, myeloid, and T cell function were stained in the indicated order and condition shown in Supplementary Table 1. Whole slide scanning was conducted by Aperio ImageScope (Leica), image processing by CellProfiler Version 2.1.1, and visualization by ImageJ Version 1.48. Image cytometry data analysis was performed by FCS Express 6 Image Cytometry Version 6.03.0011 (De Novo Software, CA, USA) based on gating strategies previously reported (13 (link)). 16 immune cell lineages including lymphoid and myeloid cell populations were quantitatively evaluated by image cytometry according to lineage selective markers shown in Supplementary Table 2.

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Tsujikawa T., Crocenzi T., Durham J.N., Sugar E., Wu A.A., Onners B., Nauroth J.M., Anders R.A., Fertig E.J., Laheru D.A., Reiss K.A., Vonderheide R.H., Ko A.H., Tempero M.A., Fisher G.A., Considine M., Danilova L., Brockstedt D.G., Coussens L.M., Jaffee E.M, & Le D.T. (2020). Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-mesothelin (CRS-207) With or Without Nivolumab in Patients with Pancreatic Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(14), 3578-3588.

Publication 2020

Aperio ImageScope FCS Express 6 Image Cytometry

Biopsies Cell function Cellularity Chromogenic Embedded paraffin Formalin Image cytometry Immunohistochemistry Lymphoid Myeloid cell Nivolumab Pathologist Populations T cell Therapy Tissue Tumor

Corresponding Organization : Sidney Kimmel Comprehensive Cancer Center

Other organizations : Oregon Health & Science University, Providence Portland Medical Center, University of Pennsylvania, University of California, San Francisco, Stanford University, Palo Alto University

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Variable analysis

independent variables

Therapy (Arm A: CY/nivolumab/GVAX and nivolumab/CRS-207, Arm B: CY/GVAX and CRS-207)

dependent variables

Immune cell lineages (lymphoid and myeloid cell populations) quantitatively evaluated by image cytometry

control variables

Tissue biopsies containing >30% tumor cellularity selected by a blinded study pathologist
Chromogenic sequential IHC conducted on 5-micron formalin-fixed paraffin-embedded tissue sections
Three 12-plex multiplex IHC panels containing markers of lymphoid, myeloid, and T cell function stained in the indicated order and condition
Image cytometry data analysis performed by FCS Express 6 Image Cytometry based on gating strategies previously reported

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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