Guidance on mucositis assessment from the MASCC Mucositis Study Group and ISOO: an international Delphi study.

Background: Mucositis is a common and highly impactful side effect of conventional and emerging cancer therapy and thus the subject of intense investigation. Although common practice, mucositis assessment is heterogeneously adopted and poorly guided, impacting evidence synthesis and translation. The Multinational Association of Supportive Care in Cancer (MASCC) Mucositis Study Group (MSG) therefore aimed to establish expert recommendations for how existing mucositis assessment tools should be used, in clinical care and trials contexts, to improve the consistency of mucositis assessment. Methods: This study was conducted over two stages (January 2022-July 2023). The first phase involved a survey to MASCC-MSG members (January 2022-May 2022), capturing current practices, challenges and preferences. These then informed the second phase, in which a set of initial recommendations were prepared and refined using the Delphi method (February 2023-May 2023). Consensus was defined as agreement on a parameter by >80% of respondents. Findings: Seventy-two MASCC-MSG members completed the first phase of the study (37 females, 34 males, mainly oral care specialists). High variability was noted in the use of mucositis assessment tools, with a high reliance on clinician assessment compared to patient reported outcome measures (PROMs, 47% vs 3%, 37% used a combination). The World Health Organization (WHO) and Common Terminology Criteria for Adverse Events (CTCAE) scales were most commonly used to assess mucositis across multiple settings. Initial recommendations were reviewed by experienced MSG members and following two rounds of Delphi survey consensus was achieved in 91 of 100 recommendations. For example, in patients receiving chemotherapy, the recommended tool for clinician assessment in clinical practice is WHO for oral mucositis (89.5% consensus), and WHO or CTCAE for gastrointestinal mucositis (85.7% consensus). The recommended PROM in clinical trials is OMD/WQ for oral mucositis (93.3% consensus), and PRO-CTCAE for gastrointestinal mucositis (83.3% consensus). Interpretation: These new recommendations provide much needed guidance on mucositis assessment and may be applied in both clinical practice and research to streamline comparison and synthesis of global data sets, thus accelerating translation of new knowledge into clinical practice. Funding: No funding was received.

Patient-reported symptom monitoring: using (big) data to improve supportive care at the macro-, meso-, and micro-levels.

PURPOSE: This paper aims to provide a comprehensive understanding of the need for continued development of symptom monitoring (SM) implementation, utilization, and data usage at the macro-, meso-, and micro-levels. METHODS: Discussions from a patient-reported SM workshop at the MASCC/ISSO 2022 annual meeting were analyzed using a macro-meso-micro analytical framework of cancer care delivery. The workshop categories "initiation and implementation, barriers to adoption and utilization, and data usage" were integrated for each level. RESULTS: At the macro-level, policy development could encourage data sharing and international collaboration, including the exchange of SM methods, supportive care models, and self-management modules. At the meso-level, institutions should adjust clinical workflow and service delivery and promote a thorough technical and clinical integration of SM. At the micro-level, SM should be individualized, with timely feedback for patients, and should foster trust and understanding of AI decision support tools amongst clinicians to improve supportive care. CONCLUSIONS: The workshop reached a consensus among international experts on providing guidance on SM implementation, utilization, and (big) data usage pathways in cancer survivors across the cancer continuum and on macro-meso-micro levels.

Baseline gut microbiota composition is associated with oral mucositis and tumour recurrence in patients with head and neck cancer: a pilot study.

PURPOSE: Mounting evidence suggests that the gut microbiome influences radiotherapy efficacy and toxicity by modulating immune signalling. However, its contribution to radiotherapy outcomes in head and neck cancer (HNC) is yet to be investigated. This study, therefore, aimed to uncover associations between an individual's pre-therapy gut microbiota and (i) severity of radiotherapy-induced oral mucositis (OM), and (ii) recurrence risk in patients with HNC. METHODS: In this prospective pilot study, 20 patients with HNC scheduled to receive radiotherapy or chemoradiotherapy were recruited. Stool samples were collected before treatment and microbial composition was analysed using 16S rRNA gene sequencing. OM severity was assessed using the NCI-CTCAE scoring system. Patients were also followed for 12 months of treatment completion to assess tumour recurrence. RESULTS: Overall, 80% of the patients were male with a median age of 65.5 years. Fifty-three percent experienced mild/moderate OM while 47% developed severe OM. Furthermore, 18% experienced tumour relapse within 1 year of treatment completion. A pre-treatment microbiota enriched of Eubacterium, Victivallis, and Ruminococcus was associated with severe OM. Conversely, a higher relative abundance of immunomodulatory microbes Faecalibacterium, Prevotella, and Phascolarctobacterium was associated with a lower risk of tumour recurrence. CONCLUSION: Our results indicate that a patient's gut microbiota composition at the start of treatment is linked to OM severity and recurrence risk. We now seek to validate these findings to determine their ability to predict treatment outcomes in HNC, with the goal of using this data to inform second-generation microbial therapeutics to optimise treatment outcomes for patients with HNC.

Oral-Gut Microbiome Axis in the Pathogenesis of Cancer Treatment-Induced Oral Mucositis.

Oral mucositis (OM) is one of the most common and debilitating oral complications of cancer treatments including chemotherapy, radiotherapy, and hematopoietic stem cell transplantation. It is associated with severe pain and difficulties in chewing, swallowing, and speech. This leads to impairment of basic oral functions and could result in unplanned treatment interruption or modification. As such, OM negatively impacts both patients' quality of life as well as tumor prognostic outcomes. Understanding pathways underlying OM pathogenesis help identify new targets for intervention or prevention. The pathophysiology of OM has been widely studied over past decades with several pathways related to oxidative stress, inflammation, and molecular and cellular signaling being implicated. In this mini-review, we will discuss the emerging role of the oral-gut microbiome axis in the development of OM. Particularly, we will elaborate on how the alterations in the oral and gut microbiota as well as intestinal dysfunction caused by cancer treatments could contribute to the pathogenesis of OM. Further, we will briefly discuss the potential methods for targeting the oral-gut microbiome axis to improve OM outcomes.

Antibiotic-Induced Gut Microbiota Depletion Accelerates the Recovery of Radiation-Induced Oral Mucositis in Rats.

PURPOSE: Due to its pivotal role in the modulation of immune and inflammatory responses, the gut microbiota has emerged as a key modulator of cancer treatment-induced gastrointestinal mucositis. However, it is not clear yet how it affects radiation therapy-induced oral mucositis (OM). As such, this study aimed to explore the gut microbiota's role in the pathogenesis of radiation-induced OM in rats. METHODS AND MATERIALS: Male Sprague Dawley rats were treated with 20 Gy x-ray radiation (Rx) delivered to the snout, with or without antibiotic-induced microbiota depletion (AIMD). OM severity was assessed, and tongue tissues were collected on day 9 and 15 postradiation for tissue injury and inflammatory markers assessment. RESULTS: AIMD+Rx had a significantly shorter duration of severe OM compared with Rx alone group. Macroscopically, the tongue ulcer-like area was smaller in AIMD+Rx compared with the Rx group. Microscopically, a smaller percentage of the mucosal ulcer was observed in the dorsal tongue of AIMD+Rx compared with the Rx group. AIMD+Rx also had significantly lower levels of interleukin 6, interleukin 1 beta, and toll like receptor 4 in the tongue tissues than the Rx group. CONCLUSIONS: The gut microbiota plays a role in OM pathogenesis, mainly in the recovery phase, through the modulation of proinflammatory pathways. Future microbiota-targeted interventions may improve OM in clinical settings.

Disparities in telehealth use: How should the supportive care community respond?

Telehealth use has increased in the setting of the COVID-19 pandemic. However, there are disparities in telehealth use based on age, income, race/ethnicity, low health, digital literacy, and limited English proficiency. There are multilevel barriers to telehealth use at the patient, health systems, telehealth portal, and policy levels. To ensure equity in telehealth services and to leverage these services to maximize the reach of health care services, concerted efforts are needed to design telehealth tools and workflows. It should include reimbursement for staff training, patient education, and technical support needed for telehealth use. Furthermore, ongoing monitoring and responsive modifications in the use of telehealth services are needed to promote telehealth equity.

Guidelines for reporting on animal fecal transplantation (GRAFT) studies: recommendations from a systematic review of murine transplantation protocols.

Fecal microbiota transplant (FMT) is a powerful tool used to connect changes in gut microbial composition with a variety of disease states and pathologies. While FMT enables potential causal relationships to be identified, the experimental details reported in preclinical FMT protocols are highly inconsistent and/or incomplete. This limitation reflects a current lack of authoritative guidance on reporting standards that would facilitate replication efforts and ultimately reproducible science. We therefore systematically reviewed all FMT protocols used in mouse models with the goal of formulating recommendations on the reporting of preclinical FMT protocols. Search strategies were applied across three databases (PubMed, EMBASE, and Ovid Medline) until June 30, 2020. Data related to donor attributes, stool collection, processing/storage, recipient preparation, administration, and quality control were extracted. A total of 1753 papers were identified, with 241 identified for data extraction and analysis. Of the papers included, 92.5% reported a positive outcome with FMT intervention. However, the vast majority of studies failed to address core methodological aspects including the use of anaerobic conditions (91.7% of papers lacked information), storage (49.4%), homogenization (33.6%), concentration (31.5%), volume (19.9%) and administration route (5.3%). To address these reporting limitations, we developed theGuidelines for Reporting Animal Fecal Transplant (GRAFT) that guide reporting standards for preclinical FMT. The GRAFT recommendations will enable robust reporting of preclinical FMT design, and facilitate high-quality peer review, improving the rigor and translation of knowledge gained through preclinical FMT studies.

Diarrhea Induced by Small Molecule Tyrosine Kinase Inhibitors Compared With Chemotherapy: Potential Role of the Microbiome.

Small molecule receptor tyrosine kinase inhibitors (SM-TKIs) are among a group of targeted cancer therapies, intended to be more specific to cancer cells compared with treatments, such as chemotherapy, hence reducing adverse events. Unfortunately, many patients report high levels of diarrhea, the pathogenesis of which remains under investigation. In this article, we compare the current state of knowledge of the pathogenesis of chemotherapy-induced diarrhea (CID) in comparison to SM-TKI-induced diarrhea, and investigate how a similar research approach in both areas may be beneficial. To this end, we review evidence that both treatment modalities may interact with the gut microbiome, and as such the microbiome should be investigated for its ability to reduce the risk of diarrhea.

Systematic review of agents for the management of cancer treatment-related gastrointestinal mucositis and clinical practice guidelines.

PURPOSE: The aim of this study was to update the clinical practice guidelines for the use of agents for the prevention and/or treatment of gastrointestinal mucositis (GIM). METHODS: A systematic review was conducted by the Mucositis Study Group of the Multinational Association of Supportive Care in Cancer/International Society for Oral Oncology (MASCC/ISOO). The body of evidence for each intervention, in each cancer treatment setting, was assigned an evidence level. Based on the evidence level, one of the following three guideline determinations was possible: Recommendation, Suggestion, and No Guideline Possible. RESULTS: A total of 78 papers across 13 interventions were examined of which 25 were included in the final review. No new guidelines were possible for any agent due to inadequate and/or conflicting evidence. Existing guidelines for probiotics and hyperbaric oxygen were unchanged. CONCLUSIONS: Of the agents studied for the prevention and treatment of GIM, the evidence continues to support use of probiotics containing Lactobacillus spp. for prevention of chemoradiotherapy and radiotherapy-induced diarrhea in patients with pelvic malignancy, and hyperbaric oxygen therapy to treat radiation-induced proctitis. Additional well-designed research is encouraged to enable a decision regarding palifermin, glutamine, sodium butyrate, and dietary interventions, for the prevention or treatment of GIM.

Gut microbiota: implications for radiotherapy response and radiotherapy-induced mucositis.

INTRODUCTION: Radiotherapy is a mainstay of solid tumor management but can be associated with unacceptable levels of off-target tissue toxicity which impact treatment outcomes and patients' quality of life. Tumour response to radiotherapy and the frequency and severity of radiotherapy-induced toxicities, especially mucositis, varies among patients. Gut microbiota has been found to modulate both the efficacy and toxicity of some types of cancer chemotherapies and immunotherapies but has yet to be investigated thoroughly in the setting of radiotherapy. Area covered: In this review, we discuss the potential role of gut microbiota on modulating radiotherapy-induced oral and gastrointestinal mucositis and the anti-tumor response to radiotherapy through modulation of immune responses. Expert opinion: The gut microbiota plays a major role in the modulation of systemic immune responses, which influence both radiotherapy response and gastrointestinal toxicities such as mucositis. Hence, investigating the gut microbiota link to the variation in radiotherapy responses and toxicities among patients is warranted. Future targeting of these responses with a patient-tailored restoration of optimal microbial composition could lead to a new era of mucositis prevention and enhanced tumor responses.

Prophylactic probiotics for cancer therapy-induced diarrhoea: a meta-analysis.

PURPOSE OF REVIEW: Strong preclinical data support prophylactic probiotics as an effective preventive strategy for diarrhoea secondary to anticancer therapies. To determine the composite evidence that this approach translates to the clinic, we performed a meta-analysis of randomized controlled trials (RCTs) of prophylactic probiotics for the prevention of cancer therapy-induced diarrhoea. RECENT FINDINGS: A three-step search strategy was used to identify relevant studies (1 June 2000-1 June 2017) investigating probiotic intervention for diarrhoea secondary to any cancer therapy (cytotoxic, targeted and immunotherapies). RCTs across PubMed, Embase, CINAHL and CENTRAL were assessed for eligibility and assessed using RevMan 5.3 (The Cochrane Collaboration). Seven trials with a total of 1091 patients were included in this meta-analysis. Compared with placebo, prophylactic probiotics did not prevent or reduce the overall incidence of diarrhoea or severe CTCAE Grade at least 3 diarrhoea [relative risk (RR) = 0.81, 95% confidence interval (95% CI) = 0.60-1.09, Z = 1.41, P = 0.16; RR = 0.54, 95% CI = 0.25-1.16, Z = 1.58, P = 0.11], nor did it influence the use of rescue medication (RR = 0.93, 95% CI = 0.53-1.65, Z = 0.24, P = 0.81). SUMMARY: Current evidence does not support widespread implementation of probiotics for diarrhoea secondary to cytotoxic therapy and the tyrosine kinase inhibitor, dacomitinib. Research efforts should be diverted to pair specific forms of gastrointestinal toxicity and their unique microbial phenotype to develop the ideal microbial protectant.

Dacomitinib-induced diarrhea: Targeting chloride secretion with crofelemer.

Dacomitinib, an irreversible small-molecule pan-ErbB TKI, has a high incidence of diarrhea, which has been suggested to be due to chloride secretory mechanisms. Based on this hypothesis, crofelemer, an antisecretory agent may be an effective intervention. T84 monolayers were treated with 1 µM dacomitinib and 10 µM crofelemer, and mounted into Ussing chambers for electrogenic ion analysis. Crofelemer attenuated increases in chloride secretion in cells treated with dacomitinib. Albino Wistar rats (n = 48) were treated with 7.5 mg/kg dacomitinib and/or 25 mg/kg crofelemer via oral gavage for 21 days. Crofelemer significantly worsened dacomitinib-induced diarrhea (p = 0.0003), and did not attenuate weight loss (p < 0.0001). Sections of the ileum and colon were mounted into Ussing chambers, and secretory processes analyzed. This indicated that crofelemer lost its anti-secretory action in the presence of dacomitinib in this model. Mass spectrometry revealed that crofelemer did not change serum concentration of dacomitinib. Serum FITC dextran levels indicated that crofelemer was unable to attenuate dacomitinib-induced barrier dysfunction. Tight junction proteins were visualized with immunofluorescence. Qualitative analysis showed dacomitinib induced proteolysis of ZO-1 and occludin, and internalization of claudin-1, which was not attenuated by crofelemer. Detailed histopathological analysis showed that crofelemer was unable to attenuate dacomitinib-induced ileal damage. Crofelemer worsened dacomitinib-induced diarrhea, suggesting that antisecretory drug therapy may be ineffective in this setting.

Selective MMP Inhibition, Using AZD3342, to Reduce Gastrointestinal Toxicity and Enhance Chemoefficacy in a Rat Model.

BACKGROUND: The common cytotoxic mechanisms that underpin chemoefficacy and toxicity have hampered efforts to deliver effective supportive care interventions, particularly for gastrointestinal (GI) toxicity. Matrix metalloproteinases (MMPs) have been implicated in both tumor growth and GI toxicity, and as such MMP inhibitors present as a novel therapeutic avenue to simultaneously enhance treatment efficacy and reduce toxicity. OBJECTIVES: The aim of this study was to determine the efficacy of an MMP-9/12 inhibitor, AZD3342, on tumor growth and GI toxicity in a rat model. METHODS: Female tumor-bearing Dark Agouti rats (n = 90) were divided into 4 groups: vehicle control; methotrexate (MTX); AZD3342, and MTX + AZD3342. Tumors were measured daily (for 5 days) using digital calipers. GI toxicity was assessed using well-established clinical markers (diarrhea/weight loss), histopathological analysis, and functional assessment of intestinal barrier permeability. RESULTS: AZD3342 delayed the onset of severe diarrhea by 1 day (vs. MTX) but was unable to improve the overall severity of diarrhea. No changes were detected in tissue morphology or intestinal barrier function. AZD3342 alone suppressed tumor growth (p = 0.003 vs. vehicle) but did not enhance the efficacy of MTX. CONCLUSIONS: This study showed partial efficacy of AZD3342 in reducing tumor growth and delaying the onset of severe diarrhea caused by MTX in rats. We suggest further studies be undertaken targeting appropriate scheduling of AZD3342 as well as investigating different cytotoxic therapies that strongly activate MMP signaling.

Dacomitinib-induced diarrhoea is associated with altered gastrointestinal permeability and disruption in ileal histology in rats.

Dacomitinib-an irreversible pan-ErbB tyrosine kinase inhibitor (TKI)-causes diarrhoea in 75% of patients. Dacomitinib-induced diarrhoea has not previously been investigated and the mechanisms remain poorly understood. The present study aimed to develop an in-vitro and in-vivo model of dacomitinib-induced diarrhoea to investigate underlying mechanisms. T84 cells were treated with 1-4 µM dacomitinib and resistance and viability were measured using transepithelial electrical resistance (TEER) and XTT assays. Rats were treated with 7.5 mg/kg dacomitinib daily via oral gavage for 7 or 21 days (n = 6/group). Weights, and diarrhoea incidence were recorded daily. Rats were administered FITC-dextran 2 hr before cull, and serum levels of FITC-dextran were measured and serum biochemistry analysis was conducted. Detailed histopathological analysis was conducted throughout the gastrointestinal tract. Gastrointestinal expression of ErbB1, ErbB2 and ErbB4 was analysed using RT-PCR. The ileum and the colon were analysed using multiplex for expression of various cytokines. T84 cells treated with dacomitinib showed no alteration in TEER or cell viability. Rats treated with dacomitinib developed severe diarrhoea, and had significantly lower weight gain. Further, dacomitinib treatment led to severe histopathological injury localised to the ileum. This damage coincided with increased levels of MCP1 in the ileum, and preferential expression of ErbB1 in this region compared to all other regions. This study showed dacomitinib induces severe ileal damage accompanied by increased MCP1 expression, and gastrointestinal permeability in rats. The histological changes were most pronounced in the ileum, which was also the region with the highest relative expression of ErbB1.

Potential safety concerns of TLR4 antagonism with irinotecan: a preclinical observational report.

PURPOSE: Irinotecan-induced gut toxicity is mediated in part by Toll-Like receptor 4 (TLR4) signalling. The primary purpose of this preclinical study was to determine whether blocking TLR4 signalling by administering (-)-naloxone, a TLR4 antagonist, would improve irinotecan-induced gut toxicity. Our secondary aim was to determine the impact of (-)-naloxone on tumour growth. METHODS: Female Dark Agouti (DA) tumour-bearing rats were randomly assigned to four treatments (n = 6 in each); control, (-)-naloxone (100 mg/kg oral gavage at -2, 24, 48, and 72 h), irinotecan (175 mg/kg intraperitoneal at 0 h), and (-)-naloxone and irinotecan. Body weight and tumour growth were measured daily, and diarrhoea incidence and severity were recorded 4× per day up to 72 h post-treatment. RESULTS: At 72 h, all rats that received irinotecan lost weight compared to controls (p = 0.03). In addition, rats that received (-)-naloxone and irinotecan lost significantly more weight compared to controls (p < 0.005) than irinotecan only compared to controls (p = 0.001). (-)-Naloxone did not attenuate irinotecan-induced severe diarrhoea at 48 and 72 h. Finally, (-)-naloxone caused increased tumour growth compared to control at 72 h (p < 0.05) and significantly reduced the efficacy of irinotecan (p = 0.001). CONCLUSIONS: (-)-Naloxone in our preclinical model was unable to block irinotecan-induced gut toxicity and decreased the efficacy of irinotecan. As (-)-naloxone-oxycodone combination is used for cancer pain, this may present a potential safety concern for patients receiving (-)-naloxone-oxycodone and irinotecan concurrently and requires further investigation.

TLR4-Dependent Claudin-1 Internalization and Secretagogue-Mediated Chloride Secretion Regulate Irinotecan-Induced Diarrhea.

We have previously shown increased intestinal permeability, to 4-kDa FITC-dextran, in BALB/c mice treated with irinotecan. Importantly, genetic deletion of Toll-like receptor 4 (TLR4; Tlr4-/-) protected against loss of barrier function, indicating that TLR4 is critical in tight junction regulation. The current study aimed (i) to determine the molecular characteristics of intestinal tight junctions in wild-type and Tlr4-/- BALB/c mice and (ii) to characterize the secretory profile of the distal colon. Forty-two female wild-type and 42 Tlr4-/- BALB/c mice weighing between 18 and 25 g received a single 270 mg/kg [intraperitoneal (i.p.)] dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72, and 96 hours. The secretory profile of the distal colon, following carbachol and forksolin, was assessed using Ussing chambers at all time points. Tight junction integrity was assessed at 24 hours, when peak intestinal permeability and diarrhea were reported, using immunofluorescence, Western blotting, and RT-PCR. Irinotecan caused internalization of claudin-1 with focal lesions of ZO-1 and occludin proteolysis in the ileum and colon of wild-type mice. Tlr4-/- mice maintained phenotypically normal tight junctions. Baseline conductance, a measure of paracellular permeability, was increased in irinotecan-treated wild-type mice at 24 hours (53.19 ± 6.46 S/cm2; P = 0.0008). No change was seen in Tlr4-/- mice. Increased carbachol-induced chloride secretion was seen in irinotecan-treated wild-type and Tlr4-/- mice at 24 hours (wild-type: 100.35 ± 18.37 µA/cm2; P = 0.022; Tlr4-/-: 102.72 ± 18.80 µA/cm2; P = 0.023). Results suggest that TLR4-dependent claudin-1 internalization and secondary anion secretion contribute to irinotecan-induced diarrhea. Mol Cancer Ther; 15(11); 2767-79. ©2016 AACR.

Cytokine-mediated blood brain barrier disruption as a conduit for cancer/chemotherapy-associated neurotoxicity and cognitive dysfunction.

Neurotoxicity is a common side effect of chemotherapy treatment, with unclear molecular mechanisms. Clinical studies suggest that the most frequent neurotoxic adverse events affect memory and learning, attention, concentration, processing speeds and executive function. Emerging preclinical research points toward direct cellular toxicity and induction of neuroinflammation as key drivers of neurotoxicity and subsequent cognitive impairment. Emerging data now show detectable levels of some chemotherapeutic agents within the CNS, indicating potential disruption of blood brain barrier integrity or transport mechanisms. Blood brain barrier disruption is a key aspect of many neurocognitive disorders, particularly those characterized by a proinflammatory state. Importantly, many proinflammatory mediators able to modulate the blood brain barrier are generated by tissues and organs that are targets for chemotherapy-associated toxicities. This review therefore aims to explore the hypothesis that peripherally derived inflammatory cytokines disrupt blood brain barrier permeability, thereby increasing direct access of chemotherapeutic agents into the CNS to facilitate neuroinflammation and central neurotoxicity.

Irinotecan-Induced Gastrointestinal Dysfunction and Pain Are Mediated by Common TLR4-Dependent Mechanisms.

Strong epidemiological data indicate that chemotherapy-induced gut toxicity and pain occur in parallel, indicating common underlying mechanisms. We have recently outlined evidence suggesting that TLR4 signaling may contribute to both side effects. We therefore aimed to determine if genetic deletion of TLR4 improves chemotherapy-induced gut toxicity and pain. Forty-two female wild-type (WT) and 42 Tlr4 null (-/-) BALB/c mice weighing between 18 and 25 g (10-13 weeks) received a single 270 mg/kg (i.p.) dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72, and 96 hours. Bacterial sequencing was conducted on cecal samples of control animals to determine the gut microbiome profile. Gut toxicity was assessed using validated clinical and histopathologic markers, permeability assays, and inflammatory markers. Chemotherapy-induced pain was assessed using the validated rodent facial grimace criteria, as well as immunologic markers of glial activation in the lumbar spinal cord. TLR4 deletion attenuated irinotecan-induced gut toxicity, with improvements in weight loss (P = 0.0003) and diarrhea (P < 0.0001). Crypt apoptosis was significantly decreased in BALB/c-Tlr4(-/-billy) mice (P < 0.0001), correlating with lower mucosal injury scores (P < 0.005). Intestinal permeability to FITC-dextran (4 kDa) and LPS translocation was greater in WT mice than in BALB/c-Tlr4(-/-billy) (P = 0.01 and P < 0.0001, respectively). GFAP staining in the lumbar spinal cord, indicative of astrocytic activation, was increased at 6 and 72 hours in WT mice compared with BALB/c-Tlr4(-/-billy) mice (P = 0.008, P = 0.01). These data indicate that TLR4 is uniquely positioned to mediate irinotecan-induced gut toxicity and pain, highlighting the possibility of a targetable gut/CNS axis for improved toxicity outcomes. Mol Cancer Ther; 15(6); 1376-86. ©2016 AACR.