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. 2023 Dec 21:13:1286754.
doi: 10.3389/fonc.2023.1286754. eCollection 2023.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma

Julyanne Brassard  1 , Michael R Hughes  1 , Pamela Dean  2 , Diana Canals Hernaez  1 , Shelby Thornton  3 , Allyson C Banville  4 , Julian Smazynski  4 , Mary Warren  4 , Kevin Zhang  4 , Katy Milne  4 , C Blake Gilks  5 , Anne-Marie Mes-Masson  6 , David G Huntsman  3   7 , Brad H Nelson  4 , Calvin D Roskelley  2 , Kelly M McNagny  1
Affiliations

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma

Julyanne Brassard et al. Front Oncol. .

Abstract

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors.

Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations.

Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome.

Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Keywords: glycoepitope; high-grade serous ovarian carcinoma; immune cold; immunotherapy; podocalyxin.

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Conflict of interest statement

Authors MH, PD, KM and CR hold patents for the PODXL antibodies described in this manuscript including issued US Patents US11090383B2, US11390673B2. KM and CR hold a patent claiming methods for detecting and treating cancer related to podocalyxin US9309323B2. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Heterogeneous expression of the PODO447 tumor glycoepitope across high-grade serous ovarian carcinoma. (A) Representative PODO447 and PODO83 immunohistochemistry (IHC) staining in HGSOC tumors. PODO447 staining was classified as negligible, moderate and high (top row) with matching cores for PODO83 staining (bottom row) (B, C) Spearman’s correlation of PODO447 and PODO83 expressing tumors presented as (B) the percentage of positive tumor cells or (C) the tumor H-score. All specimens analyzed were from naïve ovarian tumors (no-chemotherapy) in cohort A (red), B (blue), C (green) and D (black).
Figure 2
Figure 2
PODO447 tumor glycoepitope is also expressed on non-ovarian tumoral lesions in HGSOC patients. (A) PODO83 and (B) PODO447 tumor H-score in ovarian tumors (red) compared to tumors in other tissues (blue) presented as individual values and median for each patient (left panel) or mean of all the ovarian vs mean of all the tumors from other tissues for the same patient (right panel). (C) PODO83 and (D) PODO447 tumor H-score compared between ovarian tumors (red) and omentum tumors (blue) in unpaired (left panel) or paired samples (right panel). Data for AB) are from Cohort B (including naïve and post-chemotherapy samples) and data for CD) are from Cohort D. ns= non-significant; Wilcoxon (paired-data) and Mann-Witney (unpaired-data) tests.
Figure 3
Figure 3
PODO447 expression persists following chemotherapy treatment. (A) PODO83 and (B) PODO447 tumor H-score in naïve tumors (pre-chemotherapy: purple) compared to recurrent post-chemotherapy tumors (gray) presented as individual values with the median for each patient (left panel) or mean of all the naïve vs mean of all the recurrent tumors for the same patient (right panel). (C, D) PODO83 and PODO447 tumor H-score compared between naïve (purple) and post-chemotherapy (gray) in unpaired tumor samples from (C) cohort C and (D) cohort A. ns = non-significant; ****p <0.0001; Wilcoxon (paired-data) and Mann-Witney (unpaired-data) tests.
Figure 4
Figure 4
High expression of the PODO447 epitope is associated with a lower level of immune infiltrates in HGSOC. (A) Representative image of the multiplex IHC staining for CD8 (DAB), CD3 (Ferengi Blue) and CD20 (Warp Red). (B) Spearman’s correlation between PODO447 tumor H-score and the number of CD4+, CD8+ and CD3+ T cells and CD20+ cells in the stroma area (upper panel) or the tumor area (bottom panel). (C) Spearman’s correlation between PODO447 tumor H-score and the percentage of CA125, Mesothelin (MSLN) and folate receptor alpha (FOLRA) positive tumor cells. Data are from Cohort D.
Figure 5
Figure 5
Validation of the negative association between PODO447 expression and the level of CD8+ cell infiltration. (A) Representative images of the opal multiplex immunofluorescent staining used to identify CD8+ (blue) cells and CD20+ (yellow) cells in the tumor (pan-CK+) and stroma (pan-CK-) areas. (B, E) Spearman’s correlation between PODO447 tumor H-score and the number of CD8+ cells and CD20+ cells in the stroma (upper panel) or the tumor (bottom panel) areas (B) in naïve tumors and (E) in tumors that previously received NACT. (C) Scatter plot with median comparing the number of CD8+ cells and CD20+ cells in the stroma (upper panel) or in the tumor (bottom panel) areas in naïve tumors that express low (tumor H-score <5), medium (tumor H-score 5 to 50) or high level (tumor H-score >50) of the PODO447 epitope. (D) Scatter plot with median comparing the number of CD8+ cells and CD20+ cells in the stroma or in the tumor areas between tumors that previously received NACT and tumors that did not. Data are from Cohort A. * p<0.05; **p<0.01; ***p<0.001 using C: Kruskal-Wallis test and D: Mann-Whitney test. Only p-values below 0.1 are displayed.
Figure 6
Figure 6
Schematic representation of PODO447 expressing tumors. (A) Graphical representation of the low level of immune infiltration in HGSOC that expresses a high level of the PODO447 glycoepitope and representation of all the immune-phenotypes (immune-desert, immune-excluded and immune-inflamed) observed in HGSOC tumors that express low to intermediate level of the PODO447 epitope. (B) Illustration of how a PODO447-ADC could be used to turn cold PODO447-expressing tumors into hot tumors and promote immune-mediated tumor elimination. Picture created using BioRender.com.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Canadian Institutes of Health Research (PJT-166180), by a Terry Fox New Frontiers Program Project Grant and by a Canadian Cancer Society Innovation Project Grant. JB was supported by a Research Trainee Award from the Michael Smith Foundation for Health Research.