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. 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. 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. 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.

中文翻译：

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呼肠孤病毒给药后转移性结直肠癌患者的免疫特征。


KRAS 突变在 40-45% 的结直肠癌 (CRC) 患者中普遍存在，但针对该基因的治疗仍然难以捉摸。病毒是众所周知的免疫敏化剂。溶瘤呼肠孤病毒与化疗联合治疗的疗效在转移性结直肠癌的一期研究中得到检验。本研究通过确定外周循环中细胞因子的表达模式以及抗原呈递细胞 (APC) 和活化 T 淋巴细胞的分布来评估免疫反应的性质。该研究进一步评估了外泌体和细胞 microRNA 水平的变化以及呼肠孤病毒对白细胞转录组的影响。每 28 天连续 5 天静脉输注 60 分钟呼肠孤病毒，组织培养感染剂量 (TCID50) 为 3 × 1010。在呼肠孤病毒给药前和给药后从全血中分离外周血单核细胞 (PBMC)。 -第 2、8 和 15 天的呼肠孤病毒。在 EMD Millipore 多重 Luminex 平台上评估血浆中 25 种细胞因子的表达谱（PBMC 分离后）。在呼肠孤病毒处理前后的样品中测定了 miR-29a-3p 的外泌体和细胞水平。外周血单核细胞用荧光团标记的 CD4、CD8、CD56、CD70 和 CD123 抗体染色，固定并通过流式细胞术评估。颗粒酶 B 的表达是在一名患者的核心活检中测定的。最后，通过 RNA 测序分析，与呼肠孤病毒治疗前相比，Clariom D Assay 用于确定 847 个免疫相关基因的表达。如果表达水平加倍或减半，则认为存在变化，并且在 p 值为 0.001 时确定显着性。细胞因子测定表明 IL-12p40 在第 8 天上调 (2.95; p = 0.05);第 15 天，GM-CSF (3.56；p = 0.009)、IFN-y (1.86；p = 0.0004) 和 IL-12p70 (2.42；p = 0.02)。 15 天期间观察到 IL-8、VEGF 和 RANTES/CCL5 总体下降。第 15 天观察到 IL-8（0.457 倍，减少 53.3%；p = 0.03）和 RANTES/CC5（0.524 倍，减少 47.6%；p = 0.003）有统计学意义的显着减少。观察到 IL-6 总体增加，第 8 天具有统计学显着性（1.98 倍；增加 98%，p = 0.00007）。通过流式细胞术测定，APC 在 48 小时内受到刺激，并在 168 小时内激活 (CD8+ CD70+) T 细胞。据记录，外泌体和细胞中 miR-29a-3p（一种在 CRC 中上调的 microRNA，与抑癌基因 WWOX 基因表达降低相关）持续降低。 2 天后，呼肠孤病毒给药进一步导致 KRAS (33x)、IFNAR1 (20x)、STAT3(5x) 和 TAP1 (4x) 基因增加； 8 天后 FGCR2A (23x) 和 CD244 (3x)； 15 天后，KLRD1 (14x)、TAP1 (2x) 和 CD244(2x)。 2 天后观察到 VEGFA (2x) 减少 (> 0.5x)； 15 天后 CXCR2 (2x)、ITGAM (3x)。呼肠孤病毒具有深远的免疫调节特性，涵盖基因组、蛋白质和免疫细胞分布水平。这是第一项针对癌症患者的呼肠孤病毒研究，展示了这些多层次的作用，展示了呼肠孤病毒如何发挥免疫刺激剂（增强免疫化疗药物的功效）和溶瘤剂的作用。因此，呼肠孤病毒具有双模式功能，作为溶瘤剂，引起肿瘤细胞裂解，并促进免疫介导的识别和破坏肿瘤细胞。