Immune characterization of metastatic colorectal cancer patients post reovirus administration.

BACKGROUND: KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is examined in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome. METHODS: Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3 × 1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001. RESULTS: Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p = 0.05); day 15 for GM-CSF (3.56; p = 0.009), IFN-y (1.86; p = 0.0004) and IL-12p70 (2.42; p = 0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p = 0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p = 0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98- fold; 98% increase, p = 0.00007). APCs were stimulated within 48 h and activated (CD8+ CD70+) T cells within 168 h as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x), IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (> 0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days. CONCLUSIONS: Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi- layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

Oncolytic Virus Affects the RAS Pathway in Cancer: RNA Sequence Analysis.

BACKGROUND: Approximately 45% of individuals diagnosed with Colorectal Cancer (CRC) also possess KRAS mutations. One developing therapeutic method for this disease is reovirus treatment. It is theorized that reovirus treatment on patients with KRAS mutated CRC cells would be successful due to the virus' innate oncolytic properties [1]. Reovirus, a stable form of nonenveloped double-stranded RNA, causes minor infections in humans under normal circumstances. However, when the virus encounters KRAS mutated cells, it has the potential to lyse them [2]. While this method of treatment to CRC has shown signs of success, we are still some ways from universal administration of reovirus as a treatment. This review seeks to utilize various studies, as well as our original research data, to investigate reovirus as an efficient method of treatment, with a focus on select growth, apoptotic and RAS-related genes, and their effectiveness of mitigating KRAS mutated CRC post reovirus treatment. Furthermore, the review highlights transcriptome analysis as an effective tool to examine these genes and their activity. It has been shown that reovirus treatment induces apoptosis and mitigates growth related gene activity. CONCLUSIONS: This review confirms the novelty of our findings on the efficacy of reovirus in CRC treatment. The study that this review article discusses concluded that 10 apoptotic and lymphocyte-related genes were found to be upregulated and 6 angiogenesis and Ras-related genes were found to be downregulated post reovirus treatment. These findings enforce the notion that reovirus could be used as a novel treatment for KRAS mutated CRC.

Transcriptome Signature of Immune Cells Post Reovirus Treatment in KRAS Mutated Colorectal Cancer.

PURPOSE: Reovirus propagates with high efficiency in KRAS mutated colorectal cancer (CRC). About 45-50% of CRC patients possess a KRAS mutation. Oncolytic reovirus treatment in combination with chemotherapy was tested in patients possessing KRAS mutated metastatic CRC. This study evaluates the biological responses to reovirus treatment by determining the gene expression patterns in RAS-related signaling pathways. METHODS: Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3×1010. Peripheral blood mononuclear cells (PBMCs) were isolated from whole-blood pre- and post-reovirus administration at 48 hr, day-8, and day-15. Clariom_D_Human_Assay was used to determine the expression of vital genes compared to pre-reovirus treatment by RNA sequencing. Using exported sample signals, ΔΔCt method was used to analyze the fold changes of genes within seven gene pathways. Significance was calculated by students-two-tail-t-test. Hierarchical clustering dendrogram was constructed by calculating Pearson's correlation coefficients. RESULTS: As compared to the control, SOS1[48 hr; 2.49X], RRAS [48 hr; 2.24X], PIK3CB [D8, D15; 2.27X, 3.16X], MIR 16-2 [D15; 1.70X], CHORDC1 [48 hr, D15; 1.89X, 4.54X], RTN4 [48 hr; 4.66X], FAM96A [48 hr; 4.54X], NFKB [D8, D15; 19.0X, 1.42X], CASP8 [D8, D15; 2.11X, 1.77X], and CASP9 [D8; 1.45X] are upregulated post-reovirus. NOS3 [D15; 0.61X], SYNE1 [D8, D15; 0.78X, 0.71X], ANGPT1 [D8; 0.62X], VEGFB [48 hr, D8, D15; 0.44X, 0.28X, 0.28X], JUN [D15; 0.69X], and IGF2 [D8; 0.73X] are downregulated post-reovirus. Fold change values were significant [p<0.05]. CONCLUSION: This study highlights reovirus as a novel treatment option for KRAS mutated CRC and showcases its effect on the expression of crucial genes.

Oncolytic Reovirus (pelareorep) Induces Autophagy in KRAS-mutated Colorectal Cancer.

PURPOSE: To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs). EXPERIMENTAL DESIGN: HCT116 [KRAS mutant (mut)] and Hke3 [KRAS wild-type (WT)] cells were treated with pelareorep (multiplicity of infection, 5) and harvested at 6 and 9 hours. LC3 A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by Western blotting. The expression of 88 autophagy genes was determined by qRT-PCR. Syngeneic mouse models, CT26/Balb-C (KRAS mut) and MC38/C57B6 (KRAS WT), were developed and treated with pelareorep (10 × 106 plaque-forming unit/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMCs from five experimental and three control patients were sampled at 0 (pre) and 48 hours, and on days 8 and 15. The gene expression normalized to "pre" was determined using 2-ΔΔC t method. RESULTS: Pelareorep induced significant upregulation of LC3 A/B in HCT116 as compared with Hke3 cells by immunofluorescence (3.24 × and 8.67 ×), flow cytometry (2.37 × and 2.58 ×), and autophagosome formation (2.02 × and 1.57 ×), at 6 and 9 hours, respectively; all P < 0.05. Western blot analysis showed an increase in LC3 A/B (2.38 × and 6.82 ×) and Beclin1 (1.17 × and 1.24 ×) at 6 and 9 hours, ATG5 (2.4 ×) and P-62 (1.52 ×) at 6 hours, and VPS-34 (1.39 ×) at 9 hours (all P < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; P < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs (P < 0.05) was observed. LC3 A/B, RICTOR, and RASD1 expression was upregulated in all three model systems. CONCLUSIONS: Pelareorep hijacks host autophagic machinery in KRAS-mut conditions to augment its propagation and preferential oncolysis of the cancer cells.

Molecular Tools for Metastatic Colorectal Cancer Characterization.

In our recent publication [1], we have explored at the molecular level the consequences of reovirus administration to patients with KRAS mutated colorectal cancer (CRC). This was the first reported study where transcriptome assay was performed on KRAS mutated CRC patients receiving reovirus (pelareorep) therapy. Using peripheral mononuclear cells as a tumor surrogate, we have identified several hundred genes that were significantly altered in a transcriptome assay of patients receiving pelareorep serving as their own controls (pre and post reovirus administration) and compared to untreated controls [2]. We focused primarily on 884 immune related genes and published the data for genes with significance probability of 0.001 (1 in thousand for a perfect random occurrence). Samples were collected at 48 hours, day 8 and day 15 post reovirus administration and compared for dynamic gene expression alterations over time. Using PBMC we also performed flow cytometry, cytokine ELISA, immunohistochemistry, and determination of the expression level of CRC specific microRNA miR-29a-3p. Our data supports the therapeutic competence of reovirus and identifies the four major ways by which it exerts its antitumor effects.