Safety and Efficacy Outcomes in Immune Checkpoint Inhibitor-Treated Patients With Metastatic Urothelial Carcinoma Requiring Treatment Interruption or Discontinuation Due to Immune-Related Adverse Events.

INTRODUCTION: As most patients with metastatic urothelial carcinoma (mUC) will be treated with immune checkpoint inhibitors (ICI), familiarity with their associated immune-related adverse events (irAEs) is critical. We describe the characteristics and outcomes of ICI-treated mUC patients who experienced irAEs requiring treatment interruption (TI) or permanent discontinuation. MATERIALS AND METHODS: ICI-treated mUC patients who developed grade ≥2 irAEs were reviewed. Clinical-, treatment-, and toxicity-related data were evaluated. Toxicity was graded per common terminology for categorization of adverse events v5.0. Cohorts were divided into patients who underwent ICI rechallenge and those who required permanent ICI discontinuation. Time to treatment interruption (TTI), time to next treatment, and duration of clinical benefit were assessed descriptively. Progression-free survival and overall survival (OS) were estimated using Kaplan-Meier methodology. RESULTS: Of 200 ICI-treated mUC patients at Cleveland Clinic between October 2015 and October 2020, 16 (8%) experienced ≥ grade 2 irAEs necessitating TI. Median TTI among all patients was 6.5 months (range, 1-19). Eleven patients (69%) required corticosteroids. ICI were held and rechallenged in 10 patients (62%) and permanently discontinued in 6 patients (38%). Of the 10 ICI-rechallenged patients, 7 (70%) experienced another irAE upon rechallenge with median time to irAE recurrence of 2.9 months (range, 0.1-10.9); 3 (30%) eventually discontinued ICI due to recrudescent irAEs. Four (40%) of the 10 ICI-rechallenged patients received subsequent therapy. Five (83%) of the 6 patients who permanently discontinued ICI demonstrated durable clinical benefit off therapy with median duration of clinical benefit 17.7 months (range, 14.2-55.2). Two-year OS was 40% (95% CI: 19%-86%) in the ICI rechallenge cohort and 67% (95% CI: 38%-100%) in the permanent discontinuation cohort. CONCLUSION: ICI-treated mUC patients who developed irAEs requiring TI had a high rate of subsequent irAEs upon ICI rechallenge. Importantly, patients who permanently discontinued ICI due to irAE demonstrated durable clinical benefit off treatment.

Pneumonitis in Patients Receiving Thoracic Radiotherapy and Osimertinib: A Multi-Institutional Study.

Introduction: Thoracic radiotherapy (TRT) is increasingly used in patients receiving osimertinib for advanced NSCLC, and the risk of pneumonitis is not established. We investigated the risk of pneumonitis and potential risk factors in this population. Methods: We performed a multi-institutional retrospective analysis of patients under active treatment with osimertinib who received TRT between April 2016 and July 2022 at two institutions. Clinical characteristics, including whether osimertinib was held during TRT and pneumonitis incidence and grade (Common Terminology Criteria for Adverse Events version 5.0) were documented. Logistic regression analysis was performed to identify risk factors associated with grade 2 or higher (2+) pneumonitis. Results: The median follow-up was 10.2 months (range: 1.9-53.2). Of 102 patients, 14 (13.7%) developed grade 2+ pneumonitis, with a median time to pneumonitis of 3.2 months (range: 1.5-6.3). Pneumonitis risk was not significantly increased in patients who continued osimertinib during TRT compared with patients who held osimertinib during TRT (9.1% versus 15.0%, p = 0.729). Three patients (2.9%) had grade 3 pneumonitis, none had grade 4, and two patients had grade 5 events (2.0%, diagnosed 3.2 mo and 4.4 mo post-TRT). Mean lung dose was associated with the development of grade 2+ pneumonitis in multivariate analysis (OR = 1.19, p = 0.021). Conclusions: Although the overall rate of pneumonitis in patients receiving TRT and osimertinib was relatively low, there was a small risk of severe toxicity. The mean lung dose was associated with an increased risk of developing pneumonitis. These findings inform decision-making for patients and providers.

Fecal microbiota of adolescent and young adult cancer survivors and metabolic syndrome: an exploratory study.

Metabolic syndrome and obesity occur commonly in long-term pediatric cancer survivors. The intestinal microbiome is associated with metabolic syndrome and obesity in the general population, and is perturbed during cancer therapy. We aimed to determine if long-term survivors of pediatric cancer would have reduced bacterial microbiome diversity, and if these findings would be associated with components of the metabolic syndrome, obesity, and chronic inflammation. We performed a cross-sectional exploratory study examining the intestinal microbiome via 16S amplicon sequencing, treatment history, clinical measurements (blood pressure, body mass index) and biomarkers (hemoglobin A1c, lipoproteins, adiponectin: leptin ratio, C-reactive protein, TNFα, Interleukin-6, and Interleukin-10) between 35 long-term survivors and 32 age, sex, and race matched controls. All subjects were aged 10-40 years, and survivors were at least five years from therapy completion. Survivors had lower alpha diversity compared to controls (Shannon index p = .001, Simpson index p = .032) and differently abundant bacterial taxa. Further, among survivors, those who received radiation (18/35) to the central nervous system or abdomen/pelvis had decreased alpha diversity compared to those who did not receive radiation (Shannon and Simpson p < .05 for both). Although, no specific component of metabolic syndrome or cytokine was associated with measures of alpha diversity, survivors with low adiponectin-lectin ratio, elevated body mass index, and elevated C-reactive protein had differently abundant taxa compared to those with normal measures. The microbiome of cancer survivors remains less diverse than controls even many years after diagnosis, and exposure to radiation may lead to further loss of diversity in survivors.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2049937.

Treatment-free survival after discontinuation of immune checkpoint inhibitors in metastatic renal cell carcinoma: a systematic review and meta-analysis.

While immune checkpoint inhibitors (ICI) can lead to sustained responses in metastatic renal cell carcinoma (mRCC), the optimal duration of therapy remains unknown. We aimed to examine treatment-free survival (TFS) in objective responders who discontinued ICI and to explore factors that may impact objective response rate (ORR) and TFS. MEDLINE/PubMed, Embase, and the Cochrane Library were searched for prospective studies reporting individual outcomes after ICI discontinuation in patients with mRCC. Pooled ORR and TFS were estimated using random-effects meta-analyses, and associations between ICI regimen type or treatment line and ORR or TFS were evaluated. Sixteen cohorts comprising 1833 patients treated with ICI were included. The pooled ORR was 43% (95% CI 33% to 53%), and significant differences in summary estimates existed among patients who received ICI monotherapy (22%, 95% CI 18% to 26%), ICI plus a vascular endothelial growth factor (VEGF) pathway inhibitor (57%, 95% CI 48% to 65%), and dual ICI (40%, 95% CI 36% to 44%). Of 572 responders who had available data, 327 stopped ICI, with 86 (26%) continuing to respond off-treatment. Pooled TFS rates at 6 and 12 months were 35% (95% CI 20% to 50%) and 20% (95% CI 8% to 35%), respectively, and were highest for responders treated with dual ICI and lowest for those treated with ICI plus a VEGF pathway inhibitor. Thus, a subset of patients with mRCC who are treated with ICI-based therapy can have durable TFS after therapy discontinuation. Prospective clinical trials and biomarkers are needed to identify patients who can discontinue ICI therapy without compromising clinical outcomes.

A Structured Peer Assessment Method with Regular Reinforcement Promotes Longitudinal Self-Perceived Development of Medical Students' Feedback Skills.

BACKGROUND: Given that training is integral to providing constructive peer feedback, we examined the impact of a regularly reinforced, structured peer assessment method on student-reported feedback abilities throughout a two-year preclinical Communication Skills course. METHODS: Three consecutive 32-student medical school classes were introduced to the Observation-Reaction-Feedback method for providing verbal assessment during Year 1 Communication Skills orientation. In biweekly small-group sessions, students received worksheets reiterating the method and practiced giving verbal feedback to peers. Periodic questionnaires evaluated student perceptions of feedback delivery and the Observation-Reaction-Feedback method. RESULTS: Biweekly reinforcement of the Observation-Reaction-Feedback method encouraged its uptake, which correlated with reports of more constructive, specific feedback. Compared to non-users, students who used the method noted greater improvement in comfort with assessing peers in Year 1 and continued growth of feedback abilities in Year 2. Comfort with providing modifying feedback and verbal feedback increased over the two-year course, while comfort with providing reinforcing feedback and written feedback remained similarly high. Concurrently, student preference for feedback anonymity decreased. CONCLUSIONS: Regular reinforcement of a peer assessment framework can increase student usage of the method, which promotes the expansion of self-reported peer feedback skills over time. These findings support investigation of analogous strategies in other medical education settings. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40670-021-01242-w.

Interplay Between Class II HLA Genotypes and the Microbiome and Immune Phenotypes in Individuals With PTEN Hamartoma Tumor Syndrome.

We evaluate potential contributors to the development of autoimmunity and other phenotypes consistent with immune dysregulation in individuals with germline mutations in the tumor suppressor gene PTEN in this observational report. MATERIALS AND METHODS: Illumina sequencing of bacterial and fungal microbes was carried out on patient-donated fecal samples in a cohort of 67 patients with pathogenic germline PTEN mutations, including 41 individuals with autoimmunity and/or phenotypes consistent with immune dysregulation (cases) and 26 individuals without (controls). From these data, we measured differences in alpha and beta diversity between cases and controls and identified differentially abundant bacterial and fungal taxa using phyloseq and MicrobiomeSeq packages in R. We analyzed correlations between these taxa and specific HLA genotypes, along with correlations between HLA diversity and microbial diversity, by conducting high-resolution HLA genotyping at four class II loci (DRB1, DRB345, DQA1, and DQB1). RESULTS: We found that alpha diversity distributions for both bacterial and fungal genera were statistically different between cases and controls. We identified differentially abundant bacterial and fungal taxa between cases and controls. Network analysis of differentially abundant bacterial taxa revealed some co-varying bacterial genera. We additionally found significant correlations between certain HLA genotypes and certain taxa and significant correlations between HLA diversity and alpha diversity. CONCLUSION: PTEN-associated immune phenotypes might be influenced by the gut microbiome, and class II HLA molecules, in part, crosstalk with the gut microbiome. These preliminary observations should lay the groundwork for future studies to ultimately derive clinical measures, which could use gut microbiome and HLA molecule biomarkers to predict, and perhaps prevent, immunity and inflammation in patients predisposed to cancer because of germline PTEN mutations.

Human breast microbiome correlates with prognostic features and immunological signatures in breast cancer.

BACKGROUND: Currently, over half of breast cancer cases are unrelated to known risk factors, highlighting the importance of discovering other cancer-promoting factors. Since crosstalk between gut microbes and host immunity contributes to many diseases, we hypothesized that similar interactions could occur between the recently described breast microbiome and local immune responses to influence breast cancer pathogenesis. METHODS: Using 16S rRNA gene sequencing, we characterized the microbiome of human breast tissue in a total of 221 patients with breast cancer, 18 individuals predisposed to breast cancer, and 69 controls. We performed bioinformatic analyses using a DADA2-based pipeline and applied linear models with White's t or Kruskal-Wallis H-tests with Benjamini-Hochberg multiple testing correction to identify taxonomic groups associated with prognostic clinicopathologic features. We then used network analysis based on Spearman coefficients to correlate specific bacterial taxa with immunological data from NanoString gene expression and 65-plex cytokine assays. RESULTS: Multiple bacterial genera exhibited significant differences in relative abundance when stratifying by breast tissue type (tumor, tumor adjacent normal, high-risk, healthy control), cancer stage, grade, histologic subtype, receptor status, lymphovascular invasion, or node-positive status, even after adjusting for confounding variables. Microbiome-immune networks within the breast tended to be bacteria-centric, with sparse structure in tumors and more interconnected structure in benign tissues. Notably, Anaerococcus, Caulobacter, and Streptococcus, which were major bacterial hubs in benign tissue networks, were absent from cancer-associated tissue networks. In addition, Propionibacterium and Staphylococcus, which were depleted in tumors, showed negative associations with oncogenic immune features; Streptococcus and Propionibacterium also correlated positively with T-cell activation-related genes. CONCLUSIONS: This study, the largest to date comparing healthy versus cancer-associated breast microbiomes using fresh-frozen surgical specimens and immune correlates, provides insight into microbial profiles that correspond with prognostic clinicopathologic features in breast cancer. It additionally presents evidence for local microbial-immune interplay in breast cancer that merits further investigation and has preventative, diagnostic, and therapeutic potential.

Order of administration of combination cytokine therapies can decouple toxicity from efficacy in syngeneic mouse tumor models.

In combination cancer immunotherapies, consideration should be given to designing treatment schedules that harmonize with the immune system's natural timing. An efficacious temporally programmed combination therapy of extended half-life interleukin 2 (eIL2), tumor targeting antibody, and interferon (IFN) α was recently reported; however, tumor-ablative efficacy was associated with significant toxicity. In the current work, altering the order and timing of the three agents is shown to decouple toxicity from efficacy. Delaying the administration of eIL2 to be concurrent with or after IFNα eliminates toxicity without affecting efficacy in multiple syngeneic tumor models and mouse strains. The toxicity resulting from eIL2 administration before IFNα is dependent on multiple systemic inflammatory cytokines including IL6, IL10, IFNγ, and tumor necrosis factor α. Natural killer (NK) cells are the main cellular contributor to toxicity, but are not essential for tumor control in this system. When pre-conditioned with eIL2, splenic NK cells became hyper-activated and upregulate IFNα signaling proteins that cause an excessive, toxic response to subsequent IFNα exposure. This work illustrates an example where accounting for the temporal dynamics of the immune system in combination therapy treatment schedule can favorably decouple efficacy and toxicity.

Immunological Correlates of Response to Immune Checkpoint Inhibitors in Metastatic Urothelial Carcinoma.

BACKGROUND: The identification of prognostic and/or predictive biomarkers for response to immune checkpoint inhibitors (ICI) could help guide treatment decisions. OBJECTIVE: We assessed changes in programmed cell death-1 (PD1)/PD1 ligand (PDL1) expression in key immunomodulatory cell subsets (myeloid-derived suppressor cells [MDSC]; cytotoxic T lymphocytes [CTL]) following ICI therapy and investigated whether these changes correlated with outcomes in patients with metastatic urothelial carcinoma (mUC). PATIENTS AND METHODS: Serial peripheral blood samples were collected from ICI-treated mUC patients. Flow cytometry was used to quantify PD1/PDL1 expression on MDSC (CD33+HLADR-) and CTL (CD8+CD4-) from peripheral blood mononuclear cells. MDSC were grouped into monocytic (M)-MDSC (CD14+CD15-), polymorphonuclear (PMN)-MDSC (CD14-CD15+), and immature (I)-MDSC (CD14-CD15-). Mixed-model regression and Wilcoxon signed-rank or rank-sum tests were performed to assess post-ICI changes in immune biomarker expression and identify correlations between PD1/PDL1 expression and objective response to ICI. RESULTS: Of 41 ICI-treated patients, 26 received anti-PDL1 (23 atezolizumab/3 avelumab) and 15 received anti-PD1 (pembrolizumab) therapy. Based on available data, 27.5% had prior intravesical Bacillus Calmette-Guérin therapy, 42% had prior neoadjuvant chemotherapy, and 70% had prior cystectomy or nephroureterectomy. Successive doses of anti-PDL1 correlated with decreased percentage of PDL1+ (%PDL1+) M-MDSC, while doses of anti-PD1 correlated with decreased %PD1+ M- and I-MDSC. Although pre-treatment %PD1+ CTL did not predict response, a greater %PD1+ CTL within 9 weeks after ICI initiation correlated with objective response. CONCLUSIONS: Treatment with ICI correlated with distinct changes in PD1/PDL1-expressing peripheral immune cell subsets, which may predict objective response to ICI. Further studies are required to validate immune molecular expression as a prognostic and/or predictive biomarker for long-term outcomes in mUC.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

Temporally Programmed CD8α+ DC Activation Enhances Combination Cancer Immunotherapy.

Numerous synergistic cancer immunotherapy combinations have been identified, but the effects of relative dose timing are rarely considered. In established syngeneic mouse tumor models, we found that staggering interferon-α (IFNα) administration after, rather than before or simultaneously with, serum-persistent interleukin-2 (IL-2) and tumor-specific antibody significantly increased long-term survival. Successful combination therapy required IFNα-induced activation of cross-presenting CD8α+ dendritic cells (DCs) following the release of antigenic tumor debris by the IL-2- and antibody-mediated immune response. Due to decreased phagocytic ability post-maturation, DCs activated too early captured less antigen and could not effectively prime CD8+ T cells. Temporally programming DC activation to occur after tumoricidal activity enhanced tumor control by multiple distinct combination immunotherapies, highlighting dose schedule as an underappreciated factor that can profoundly affect the success of multi-component immunotherapies.

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

Protein Engineering and Selection Using Yeast Surface Display.

Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution.

Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody-cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R-expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibody-antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.

Treating periprosthetic joint infections as biofilms: key diagnosis and management strategies.

Considerable evidence suggests that microbial biofilms play an important role in periprosthetic joint infection (PJI) pathogenesis. Compared to free-floating planktonic bacteria, biofilm bacteria are more difficult to culture and possess additional immune-evasive and antibiotic resistance mechanisms, making infections harder to detect and eradicate. This article reviews cutting-edge advances in biofilm-associated infection diagnosis and treatment in the context of current PJI guidelines and highlights emerging technologies that may improve the efficacy and reduce costs associated with PJI. Promising PJI diagnostic tools include culture-independent methods based on sequence comparisons of the bacterial 16S ribosomal RNA gene, which offer higher throughput and greater sensitivity than culture-based methods. For therapy, novel methods based on disrupting biofilm-specific properties include quorum quenchers, bacteriophages, and ultrasound/electrotherapy. Since biofilm infections are not easily detected or treated by conventional approaches, molecular diagnostic techniques and next-generation antibiofilm treatments should be integrated into PJI clinical practice guidelines in the near future.

Evaluation and management of metal hypersensitivity in total joint arthroplasty: a systematic review.

Metal hypersensitivity has been an identified problem in orthopedics for nearly half a century, but its implications remain unclear. Establishing which total joint arthroplasty (TJA) candidates may do poorly with conventional implants and which patients would benefit from revision to an allergen-free implant remains challenging. Our systematic search of the MEDLINE database identified 52 articles for inclusion in our review. Case reports revealed that half of patients presented with pain and swelling, while only one-third presented with cutaneous symptoms. All patients were symptomatic within the first post-operative year; 90% were symptomatic within 3 months. Reports of patch testing revealed that patients with TJAs were positive for metal sensitivity more often than patients without TJAs (OR 1.3). Those with poorly functioning arthroplasties and those who had already had revisions tested positive more often than those with well-functioning TJAs (OR 1.7) and those without TJAs (OR 3.1). Lymphocyte transformation testing (LTT) shows promise in diagnosing metal allergy, and components of bone cement are also being recognized as potential allergens. Further work is necessary to delineate which patients should be tested for metal allergy and which patients would benefit from allergen-free implants.

The intestinal flora is required to support antibody responses to systemic immunization in infant and germ free mice.

The presence of a complex and diverse intestinal flora is functionally important for regulating intestinal mucosal immune responses. However, the extent to which a balanced intestinal flora regulates systemic immune responses is still being defined. In order to specifically examine whether the acquisition of a less complex flora influences responses to immunization in the pre-weaning stages of life, we utilize a model in which infant mice acquire an intestinal flora from their mothers that has been altered by broad-spectrum antibiotics. In this model, pregnant dams are treated with a cocktail of antibiotics that alters both the density and microbial diversity of the intestinal flora. After challenge with a subcutaneous immunization, the antibiotic altered flora infant mice have lower antigen specific antibody titers compared to control age-matched mice. In a second model, we examined germ free (GF) mice to analyze how the complete lack of flora influences the ability to mount normal antibody responses following subcutaneous immunization. GF mice do not respond well to immunization and introduction of a normal flora into GF mice restores the capacity of these mice to respond. These results indicate that a gastrointestinal flora reduced in density and complexity at critical time points during development adversely impacts immune responses to systemic antigens.