Hyperthermic Intraperitoneal Chemotherapy-Induced Molecular Changes in Humans Validate Preclinical Data in Ovarian Cancer

Abstract

Purpose: Hyperthermic intraperitoneal chemotherapy (HIPEC) confers a survival benefit in epithelial ovarian cancer (EOC) and in preclinical models. However, the molecular changes induced by HIPEC have not been corroborated in humans.

Patients and methods: A feasibility trial evaluated clinical and safety outcomes of HIPEC with cisplatin during optimal cytoreductive surgery (CRS) in patients with EOC diagnosed with stage III, IV, or recurrent EOC. Pre- and post-HIPEC biopsies were comprehensively profiled with genomic and transcriptomic sequencing to identify mutational and RNAseq signatures correlating with response; the tumor microenvironment was profiled to identify potential immune biomarkers; and transcriptional signatures of tumors and normal samples before and after HIPEC were compared to investigate HIPEC-induced acute transcriptional changes.

Results: Thirty-five patients had HIPEC at the time of optimal CRS; all patients had optimal CRS. The median progression-free survival (PFS) was 24.7 months for primary patients and 22.4 for recurrent patients. There were no grade 4 or 5 adverse events. Anemia was the most common grade 3 adverse event (43%). Hierarchical cluster analyses identified distinct transcriptomic signatures of good versus poor responders to HIPEC correlating with a PFS of 29.9 versus 7.3 months, respectively. Among good responders, significant HIPEC-induced molecular changes included immune pathway upregulation and DNA repair pathway downregulation. Within cancer islands, % programmed cell death protein 1 expression in CD8+ T cells significantly increased after HIPEC. An exceptional responder (PFS 58 months) demonstrated the highest programmed cell death protein 1 increase. Heat shock proteins comprised the top differentially upregulated genes in HIPEC-treated tumors.

Conclusion: Distinct transcriptomic signatures identify responders to HIPEC, and preclinical model findings are confirmed for the first time in a human cohort.

Trial registration: ClinicalTrials.gov NCT01970722.

Conflict of interest statement

Ernest S. HanResearch Funding: Vergent Bioscience (Inst) Mustafa RaoofOpen Payments Link: https://openpaymentsdata.cms.gov/physician/3634542 Saul J. PricemanStock and Other Ownership Interests: Imugene Ltd, Adicet BioConsulting or Advisory Role: Imugene Ltd, Adicet Bio, MustangBio, BayerResearch Funding: Imugene Ltd, Carisma TherapeuticsPatents, Royalties, Other Intellectual Property: CAR Therapeutic, Oncolytic Virus Therapeutic Jeff F. LinHonoraria: C-SATSConsulting or Advisory Role: Aspira Women's HealthTravel, Accommodations, Expenses: Intuitive Surgical, GlaxoSmithKline Mehdi KebriaHonoraria: GlaxoSmithKlineSpeakers' Bureau: GlaxoSmithKline Nora RuelConsulting or Advisory Role: EPHOS Bioscience Benjamin I. PazEmployment: City of HopeStock and Other Ownership Interests: Bio-Path Holdings Inc Mark T. WakabayashiEmployment: RegeneronStock and Other Ownership Interests: Regeneron Mihaela C. CristeaHonoraria: AstraZeneca, AbbVieConsulting or Advisory Role: AstraZeneca, AbbVieSpeakers' Bureau: AstraZenecaNo other potential conflicts of interest were reported.

Figures

FIG 1.

Study schema: (A) study flow and (B) flow chart of data processing and analysis. HIPEC, hyperthermic intraperitoneal chemotherapy; PFS, progression-free survival.

FIG 2.

Patient outcomes (survival and safety): (A) Swimmer plot showing disease status and outcomes for all patients with a follow-up of 72 months. (B) Kaplan-Meier survival curve depicting PFS and OS for all patients. (C) AEs—treatment-related toxicity. Bars represent on-study and follow-up period. AE, adverse event; NR, not reached; OS, overall survival; PFS, progression-free survival.

FIG 3.

Outcome-related gene and mutational signatures of HIPEC responders. (A) Hierarchical clustered analysis of significantly changed genes in tumor samples of 15 patients. Responders were categorized into good responders (PFS > 12 months) and poor responders (PFS

FIG 4.

Tumor microenvironment changes induced by HIPEC. Multiplex immunofluorescence estimation of tumor-infiltrating immune subsets and PD-1 expression in matched pre- and post HIPEC tumors. (A) Pre- and post-HIPEC tumors were stained with TIL markers (CD3, CD8, FOXP3), PD-1, for the following phenotypes: CD4+ conventional T cells (CD3+ CD8– FOXP3– cells), CD4+ regulatory T cells (CD3+ CD8– FOXP3+), and CD8+ T cells (CD3+ CD8+). (B) Cell density of CD8+ T cells, CD4+ conventional T cells, and CD4+ regulatory T cells do not change with HIPEC treatment. (C) Evaluation of PD-1 expression in CD8+ T cells. PD-1 expression rises in CD8+ T cells within cancer islands (cancer), but not in stroma. (D) Representative immunofluorescence demonstrating increased PD-1 staining of CD8+ T cells within cancer islands after HIPEC while stromal PD-1 expression remained stable after HIPEC. (E) CK was used as a marker for cancer islands to delineate it from stroma. (F) PD-1 expression changes in individual patients before and after HIPEC in CD8+ T cells (stroma). The patient with the highest rise in %PD-1 was patient 1, an exceptional responder with PFS of 5 years and demonstrated the largest PD-1 increase. The only patient with decreased PD-1 expression was patient 8, who was the only patient with clear cell histology and had a poor survival (PFS 24 months) and poor (PFS

FIG 5.

Gene and pathway changes of pre- and post-HIPEC in metastatic tumor and normal patient samples. (A and B) Hierarchical clustered analysis of (A) differentially expressed genes in metastatic tumors and (B) normal samples. Cluster 1 was enriched for pre-HIPEC; cluster 2 was enriched with post-HIPEC. (C) Volcano plot displaying gene expression post-HIPEC by log2-fold change (x-axis) and minus log10P value (y-axis) in tumor samples. Significantly upregulated genes of ≥ 2-fold expression are in red while statistically significantly downregulated genes of ≥ 2-fold expression are in green. The top five differentially expressed genes with fold change (post- v pre-HIPEC) and P value shown for tumor samples are shown. (D) Volcano plot displaying gene expression post-HIPEC by log2-fold change (x-axis) and minus log10P value (y-axis) in normal samples. (E and F) Kegg pathway gene set enrichment analysis of (E) HIPEC metastatic tumors and (F) normal samples demonstrating the top significantly upregulated and downregulated pathways (FDR < 0.05). Several immune-related pathways are upregulated, and DNA replication pathways are downregulated in tumor samples. Normal HIPEC samples demonstrate significant downregulation of metabolic pathways. FC, fold change; FDR, false discovery rate; HIPEC, hyperthermic intraperitoneal chemotherapy; NES, normalized enrichment score; q. val., q value; TCA, tricarboxylic acid.