Letter to the Editor: Reovirus as an immunomodulatory adjuvant in KRAS-mutant colorectal cancer - translational insights and future directions

Abstract

The recent study by Zweig et al published in the World Journal of Clinical Oncology highlights the promising role of reovirus in modulating key signaling pathways and cytokine profiles in metastatic colorectal cancer patients with KRAS mutations. Their findings demonstrate significant alterations in gene expression (e.g., signal transducer and activator of transcription 3, glycogen synthase kinase 3 beta) and cytokine dynamics (e.g. vascular endothelial growth factor suppression, interferon-gamma elevation), suggesting reovirus-induced antitumor immunity. While these results underscore reovirus’s potential as a therapeutic adjuvant, critical questions remain regarding its long-term efficacy, optimal dosing, and synergy with existing therapies. This letter discusses the translational implications of these findings, emphasizing the need for larger clinical cohorts, tumor microenvironment-focused analyses, and combinatorial strategies with immune checkpoint inhibitors to maximize therapeutic impact. Addressing these gaps could accelerate reovirus’s integration into personalized oncology regimens for KRAS-driven malignancies.

Key Words: Reovirus; KRAS mutation; Immune modulation; Tumor microenvironment; Metastatic colorectal cancer; Signaling pathway; Cytokine

Core Tip: This study discussed that reovirus affects gene expression and cytokine secretion by regulating phosphatidylinositol 3-kinase/protein kinase B and other signaling pathways, thereby regulating the immune system and ultimately exerting an anti-KRAS mutant colon cancer effect. The letter proposes to more accurately elucidate the local immune regulation mechanism of reovirus through large-scale, multi-center randomized controlled trials, combined with tumor microenvironment analysis and combined immunotherapy design, and enrich experimental analysis methods, laying a good foundation for clinical application of reovirus.

TO THE EDITOR

The recent study by Zweig et al[1] published in the World Journal of Clinical Oncology, represents a timely exploration of reovirus (pelareorep) as an oncolytic platform for KRAS-mutant metastatic colorectal cancer (mCRC). In the evolving landscape of cancer immunotherapy, oncolytic viruses have emerged not merely as cytolytic agents but as potent immunomodulatory adjuvants capable of “heating up” cold tumors[2,3]. This is particularly critical for KRAS-mutant mCRC, a subtype notoriously resistant to epidermal growth factor receptor (EGFR) inhibitors and characterized by a metabolic and immunosuppressive tumor microenvironment (TME) that facilitates immune evasion[4-7].

The innovation of Zweig et al[1] lies in their systematic profiling of the host immune landscape post-reovirus administration. By integrating transcriptomics and enzyme-linked immunosorbent assay, the authors highlighted a sophisticated dual-action mechanism: Direct viral oncolysis and systemic immune remodeling[8]. Specifically, the observed downregulation of tumor-promoting signaling (e.g., signal transducer and activator of transcription 3, glycogen synthase kinase 3 beta) and the favorable shift in cytokine dynamics - marked by the suppression of pro-angiogenic vascular endothelial growth factor (VEGF) and the elevation of anti-tumor interferon-gamma (IFN-γ) - align with the current paradigm of virally-induced “immuno-sensitization”[9]. This mechanistic rationale is further supported by evidence that intravenous delivery of reovirus can immunologically prime patients for subsequent checkpoint blockade[10], a clinical frontier of intense investigation.

However, despite these provocative findings, several critical methodological and interpretive limitations merit further discussion. First, the statistical power of the study is constrained by the limited sample size (n = 5 in the treatment cohort). Given the substantial inter-patient heterogeneity in mCRC, such a limited cohort may fail to capture the universal biological impact of the therapy. Second, a significant “biological gap” exists between peripheral signals and the actual TME. The reliance on bulk peripheral blood mononuclear cell transcriptomics assumes that circulating immune signatures serve as faithful surrogates for the metastatic niche. However, as underscored by the need for high-dimensional immune-profiling in modern oncology[11], the true complexity of TME remodeling - including the crosstalk between pyroptosis and cytokine release[12] - often eludes bulk peripheral analysis.

Furthermore, the study’s mechanistic rigor is weakened by the absence of functional validation and data inconsistencies, such as the missing V-Akt murine thymoma viral oncogene homolog 1 graph line in Figure 4A. More importantly, the lack of correlation between the observed transcriptomic shifts and definitive clinical endpoints (e.g., progression-free survival or Response Evaluation Criteria in Solid Tumors-defined regression) leaves the clinical translation of these findings speculative. In summary, while Zweig et al[1] provide a valuable molecular snapshot, future studies must incorporate larger cohorts and high-resolution techniques to confirm that these peripheral “molecular echoes” truly reflect a durable intratumoral therapeutic impact.

REOVIRUS: SELECTIVE ONCOLYSIS AND IMMUNE REPROGRAMMING IN KRAS-MUTANT CRC

EGFR inhibitors, such as cetuximab and panitumumab, primarily block upstream signaling through the EGFR. In contrast, KRAS mutations occur at a critical downstream node within the RAS-RAF-MEK-ERK (mitogen-activated protein kinase) pathway. Once mutated, KRAS remains constitutively active (predominantly in the guanosine triphosphate-bound state), thereby bypassing EGFR inhibition and directly driving key oncogenic processes, including cell proliferation, survival, and metastasis[13]. Consequently, KRAS mutations - present in approximately 40%-50% of colorectal cancers - serve as negative predictive biomarkers for anti-EGFR therapy[14]. Patients harboring these mutations derive little to no clinical benefit from EGFR inhibitors and, in some cases, may even experience detrimental effects[15].

Reovirus infection involves the release of double-stranded RNA (dsRNA) following entry into tumor cells. In normal cells, dsRNA triggers activation and autophosphorylation of protein kinase R, which in turn phosphorylates eukaryotic translation initiation factor 2α. This leads to suppression of host protein synthesis, inhibition of viral mRNA translation and replication, and rapid clearance of the virus. In KRAS-mutant tumor cells, however, persistent activation of the RAS signaling pathway - particularly via the MEK/ERK axis - strongly suppresses protein kinase R phosphorylation. As a result, eukaryotic translation initiation factor 2α remains largely unphosphorylated, permitting efficient translation of viral proteins, robust viral genome replication, and the formation of cytoplasmic viral factories[16,17]. This ultimately results in the production of large numbers of progeny virions, induction of cancer cell apoptosis (evidenced by activation of caspase-3, poly (adenosine diphosphate-ribose) polymerase cleavage, and other markers), and subsequent lysis, which releases virions to infect neighboring tumor cells[18].

In patients with KRAS-mutant colorectal cancer (and other malignancies), preclinical and clinical studies demonstrate that reovirus infection of tumor cells releases dsRNA, which engages pattern recognition receptors such as Toll-like receptor 3, retinoic acid-inducible gene I, and melanoma differentiation-associated protein 5. This activates downstream signaling through nuclear factor kappa B and interferon regulatory factor 3 pathways, culminating in the production and secretion of a broad array of cytokines and chemokines. Pro-inflammatory cytokines - including interleukin-1β, interleukin-6, tumor necrosis factor-alpha, type I and II interferons (IFN-α/β/γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) - are markedly upregulated[19]. These mediators promote dendritic cell maturation, natural killer cell activation[20], and indirect stimulation of T-cell expansion and recruitment, particularly of CD8+ cytotoxic T lymphocytes, including those specific for KRAS-mutant neoantigens[11]. Concurrently, increased secretion of chemokines such as C-C motif ligands 4 and others facilitates the recruitment of immune effector cells into the TME and enhances the infiltration and reactivation of circulating tumor-infiltrating lymphocytes, thereby augmenting antitumor immunity and facilitating immune-mediated tumor cell killing[11,19].

During reovirus treatment, dynamic changes in cytokine profiles significantly influence the TME. Notably, the elevation of IFN-γ serves as a key marker of natural killer cell activation and the expansion of Th1-polarized CD8+ T cells. This creates a favorable immunological window, particularly between days 8 and 15 post-treatment (corresponding to the peak of adaptive immune activation), during which sequential or concurrent administration of programmed cell death protein 1 (PD-1)/programmed death-ligand 1 inhibitors may substantially amplify antigen-specific adaptive immunity and help convert the immunologically “cold” state characteristic of microsatellite-stable (MSS) tumors[21].

Translational studies have further demonstrated expansion of KRAS-mutant-specific T-cell clones following reovirus exposure, underscoring its potential to elicit tumor antigen-specific adaptive responses in KRAS-driven malignancies[21,22]. In parallel, the observed suppression of VEGF directly attenuates tumor angiogenesis and vascular permeability. This effect can be leveraged through sequential or simultaneous combination with bevacizumab (or other anti-VEGF agents) to promote vascular normalization, thereby enhancing immune cell infiltration and synergistic antitumor activity[21,23,24].

In the phase 1 REO 022 trial evaluating pelareorep in combination with bevacizumab plus folinic acid, fluorouracil, and irinotecan in patients with KRAS-mutant MSS mCRC, an objective response rate of 33% was achieved - substantially exceeding the historical control range of 6%-11% observed with standard second-line regimens. Moreover, the regimen was associated with a median progression-free survival of 16.6 months and a median overall survival of 27.0 months, representing marked improvements over historical benchmarks[21].

Compared with talimogene laherparepvec (herpes simplex virus type1-based and engineered to express GM-CSF), reovirus activates the Toll-like receptor 3/retinoic acid-inducible gene I/nuclear factor kappa B pathway via its natural dsRNA genome, without requiring genetic modification. This enables broader innate immune reprogramming, including dendritic cell maturation, indirect natural killer cell activation, and induction of a diverse pro-inflammatory cytokine response. In contrast, talimogene laherparepvec primarily relies on local GM-CSF expression to recruit dendritic cells and promote systemic T-cell memory, rendering it more suitable for intratumoral injection in cutaneous or accessible metastatic melanoma[19,25].

Relative to adenoviruses (typically requiring E1A/E1B deletions or other engineering), reovirus exhibits superior tolerability for systemic intravenous administration, with minimal hepatotoxicity, limited impact from pre-existing neutralizing antibodies, and inherent selectivity for RAS/KRAS-activated tumors. Although adenoviruses can accommodate various transgenes, they frequently elicit strong antiviral immunity that restricts repeated dosing[19,24,26].

Consequently, reovirus is uniquely positioned as a natural immune-reprogramming platform for RAS pathway-dependent “cold” tumors, particularly in refractory subgroups such as KRAS-mutant MSS colorectal cancer and pancreatic cancer, where options remain limited after EGFR inhibitor resistance. Its core advantages - mechanism-driven selective oncolysis combined with broad-spectrum cytokine induction - facilitate seamless integration with chemotherapy, anti-VEGF agents, immune checkpoint inhibitors, or emerging KRAS G12C inhibitors, establishing it as a safe, accessible, and highly translatable strategy in oncolytic virotherapy[19].

MAJOR LIMITATIONS

This study has some obvious limitations. First, the sample size of the included patients is too small (only 5 cases), which significantly limits the statistical robustness and extrapolation ability of the results, and the influence of individual variation and random error may be amplified. Secondly, the study lacks long-term follow-up data for patients, and fails to provide key clinical endpoint information such as progression-free survival and overall survival, which limits the evaluation of efficacy persistence and long-term safety. It is recommended that follow-up studies should significantly expand the sample size, adopt a multi-center, prospective cohort design, and systematically report the long-term survival outcomes and quality of life indicators of patients.

METHODOLOGICAL SHORTCOMINGS

In terms of methodology, this study mainly relies on peripheral blood samples for cytokine analysis. Although it is easy to operate and has certain reference value, there is a certain degree of decoupling between peripheral blood immune status and TME, and peripheral indicators cannot fully represent tumor local immune response[6,27]. Therefore, the tumor biological significance of the obtained cytokine changes has certain limitations. Future studies should combine tumor biopsy samples to carry out flow cytometry, single-cell RNA sequencing or multiple immunofluorescence analysis to more directly and comprehensively characterize the changes[28], functional status and spatial distribution of immune cell subsets in the TME, so as to more accurately elucidate the local immune regulation mechanism of reovirus.

MORE CAUTIOUS INTERPRETATION OF RESULTS AND POTENTIAL RISKS

In addition, the interpretation of some results may be optimistic. The error bars of multiple sets of data such as Figure 2A and B, Figure 3A and Figure 4A are relatively large, suggesting significant intra-group variation. The statistical significance and clinical relevance need to be carefully weighed. For example, Figure 2B shows that the decrease of VEGF level is 20%-36%, and the change range is relatively limited, and its clinical significance remains to be further verified. At the same time, the study did not fully address the potential risk of adverse reactions (such as excessive immune activation or inflammatory storms), which is particularly important in oncolytic virus therapy[8]. Combined with the absence of other oncolytic viruses or immune checkpoint inhibitors in this study, it is difficult to judge the relative efficacy advantage of reovirus in this population. In the future, it is advisable to carry out head-to-head comparative studies and actively explore the combined application of reovirus with PD-1/programmed death-ligand 1 inhibitors, cytotoxic T-lymphocyte-associated protein 4 inhibitors or other immunomodulatory strategies in order to achieve synergies[3].

TRANSLATIONAL INSIGHTS AND FUTURE DIRECTIONS

To address the challenge of the “immune-cold” TME in KRAS-mutated MSS mCRC, we hypothesize that pelareorep can reverse this immunosuppressive state through two complementary mechanisms: (1) Elevation of peripheral IFN-γ levels, thereby promoting tumor infiltration of KRAS-specific CD8+ T cells; and (2) Modulation of the hosphatidylinositol 3-kinase/protein kinase B pathway (upregulation of phosphatase and tensin homolog pseudogene 1 and downregulation of rapamycin-insensitive companion of mammalian target of rapamycin), leading to VEGF suppression, vascular normalization, and enhanced recruitment of effector immune cells.

On the basis of this dual-mechanism hypothesis, we propose a multicenter, biomarker-stratified, Simon two-stage phase II trial (n = 40-60) enrolling patients with KRAS-mutated (prioritizing G12/G13 variants) MSS mCRC who have progressed after second-line therapy and have an Eastern Cooperative Oncology Group performance status of 0-1. The investigational regimen consists of pelareorep (3 × 10¹⁰ median tissue culture infectious dose 50/day on days 1-5 of each 28-day cycle) combined with folinic acid, fluorouracil, and irinotecan plus bevacizumab, with or without a PD-1 inhibitor. The primary endpoint is the rate of TME immune “hot” conversion (defined as > 50% of patients shifting from a cold to a hot phenotype, assessed by single-cell RNA sequencing and complementary platforms). We anticipate an objective response rate of ≥ 35%, substantially exceeding the historical benchmark of approximately 15%. Mechanism validation will be performed through mandatory paired tumor biopsies, spatial transcriptomics, and multiplex immunofluorescence, providing robust pharmacodynamic evidence to support advancement to a confirmatory phase III study. The following streamlined hypotheses and designs are proposed (Table 1).

Table 1 Proposed testable hypotheses and designs to overcome peripheral blood limitations.

Core hypothesis (key question)	Proposed design (main steps)	Expected outcomes
Does peripheral blood IFN-γ elevation correspond to enhanced infiltration of KRAS-mutant-specific CD8+ T cells in the TME, thereby reversing the immunologically cold state in MSS-type CRC?	Multicenter cohort (n > 50, KRAS-mutant mCRC); Matched peripheral blood + tumor biopsies (baseline, days 8/15); scRNA-seq + TCR-seq (IFN-γ gene signatures vs KRAS-specific clones); Flow cytometry to detect CD8+PD-1+ cells	Correlation coefficient > 0.7 (peripheral IFN-γ vs TME T-cell metrics), confirming immune hot conversion
Does reovirus (pelareorep) regulate the PI3K/AKT pathway (phosphatase and tensin homolog pseudogene 1 increase, rapamycin-insensitive companion of mammalian target of rapamycin decrease) to suppress VEGF expression in the TME, leading to vascular normalization and increased immune cell recruitment?	KRAS-mutant PDX models + clinical biopsies; multiplex immunofluorescence (co-localization of VEGF/PI3K with CD31+ vessels); scRNA-seq (endothelial subpopulation changes and immune scores)	> 30% reduction in VEGF expression post-treatment, accompanied by increased TME immune scores and vascular normalization
Biomarker-driven phase II trial to validate TME immune conversion and efficacy in KRAS-mutant MSS mCRC	Multicenter Simon two-stage phase II trial (n = 40-60); patient population: KRAS-mutant (prioritizing G12/G13 subtypes), MSS mCRC, after second-line failure, Eastern Cooperative Oncology Group 0-1; stratification: Baseline TME signature (high/Low VEGF; low/high IFN-γ); intervention: Pelareorep (3 × 1010 tissue culture infectious dose 50/day × 5 days, every 28 days) + folinic acid, fluorouracil, and irinotecan + bevacizumab ± PD-1 inhibitor; primary endpoint: TME immune conversion rate (> 50% shift to “hot” phenotype, assessed by scRNA-seq); biomarkers: Mandatory biopsies + spatial transcriptomics (e.g., GeoMx) to monitor PI3K/AKT heterogeneity and IFN-γ signatures; statistics: Kaplan-Meier survival analysis + Wilcoxon paired test (80% power, α = 0.05)	Confirmation of TME immune hot conversion, ORR ≥ 35% (vs historical approximately 15%), improved progression-free survival, supporting advancement to phase III

IFN: Interferon-gamma; TME: Tumor microenvironment; MSS: Microsatellite stable; CRC: Colorectal cancer; mCRC: Metastatic colorectal cancer; scRNA-seq: Single-cell RNA sequencing; TCR-seq: T-cell receptor sequencing; PD-1: Programmed cell death protein 1; PI3K/AKT: Phosphatidylinositol 3-kinase/protein kinase B; VEGF: Vascular endothelial growth factor; PDX: Patient-derived xenograft; ORR: Objective response rate.

CONCLUSION

This study innovatively explored the application of reovirus in KRAS mutant mCRC, and revealed its dual mechanism through transcriptomics and enzyme-linked immunosorbent assay: Directly killing tumor cells and regulating key pathways to reshape the immune landscape, providing a preliminary conceptual verification and treatment perspective for refractory subtypes, which has certain potential. However, the limitations of small sample size (only 5 cases), lack of long-term survival data, relying only on peripheral blood indicators, large statistical variation, no control group and adverse reaction discussion make the evidence strength seriously insufficient, which is only a highly preliminary exploration. In the future, more large-scale, multi-center randomized controlled trials are needed, combined with TME analysis and combined immunotherapy design, in order to truly verify its efficacy and clinical value.