Two faces: IL-22 effects prevail over defense against metastasis.

Interleukin-22 (IL-22) is a cytokine with pleotropic and opposing roles in physiological and pathological states. In this issue of Immunity, Giannou et al. and Briukhovetska et al. demonstrate how IL-22 is involved in promoting cancer metastasis formation.

Cancer cell-intrinsic mechanisms driving acquired immune tolerance.

Immune evasion is a hallmark of cancer, enabling tumors to survive contact with the host immune system and evade the cycle of immune recognition and destruction. Here, we review the current understanding of the cancer cell-intrinsic factors driving immune evasion. We focus on T cells as key effectors of anti-cancer immunity and argue that cancer cells evade immune destruction by gaining control over pathways that usually serve to maintain physiological tolerance to self. Using this framework, we place recent mechanistic advances in the understanding of cancer immune evasion into broad categories of control over T cell localization, antigen recognition, and acquisition of optimal effector function. We discuss the redundancy in the pathways involved and identify knowledge gaps that must be overcome to better target immune evasion, including the need for better, routinely available tools that incorporate the growing understanding of evasion mechanisms to stratify patients for therapy and trials.

Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4+ T Cells.

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.

Neoantigen-directed immune escape in lung cancer evolution.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

REFINE-Lung implements a novel multi-arm randomised trial design to address possible immunotherapy overtreatment.

Increasing evidence suggests that some immunotherapy dosing regimens for patients with advanced cancer could result in overtreatment. Given the high costs of these agents, and important implications for quality of life and toxicity, new approaches are needed to identify and reduce unnecessary treatment. Conventional two-arm non-inferiority designs are inefficient in this context because they require large numbers of patients to explore a single alternative to the standard of care. Here, we discuss the potential problem of overtreatment with anti-PD-1 directed agents in general and introduce REFINE-Lung (NCT05085028), a UK multicentre phase 3 study of reduced frequency pembrolizumab in advanced non-small-cell lung cancer. REFINE-Lung uses a novel multi-arm multi-stage response over continuous interventions (MAMS-ROCI) design to determine the optimal dose frequency of pembrolizumab. Along with a similarly designed basket study of patients with renal cancer and melanoma, REFINE-Lung and the MAMS-ROCI design could contribute to practice-changing advances in patient care and form a template for future immunotherapy optimisation studies across cancer types and indications. This new trial design is applicable to many new or existing agents for which optimisation of dose, frequency, or duration of therapy is desirable.

Novel approaches to managing gestational trophoblastic tumors in the age of immunotherapy.

The discovery that anti-programmed death-1 antibody (anti-PD-1) immunotherapy can cure patients with multidrug-resistant gestational trophoblastic neoplasia provides a new powerful and low toxicity treatment. This heralds an era within which the majority of patients, including those with previously difficult to treat disease, can expect to achieve long-term remission. This development should prompt a rethink of how patients with this rare disease are managed, focusing on maximizing cure rate with minimal exposure to toxic chemotherapy.

Immunotherapy for GTN: a new paradigm.

Background Immune checkpoint immunotherapy (CPI) targeting PD1/PD-L1 has been shown to be an effective treatment for gestational trophoblastic neoplasia (GTN). This includes those with multidrug resistance, ultra-high risk disease and ETT/PSTT subtypes that are inherently chemotherapy resistant, but there is also emerging evidence in low-risk disease. Objectives We set out to generate an overview of the current data supporting the use of CPI for GTN in both high risk and low risk disease and to consider future research goals and directions in order to implement CPI in current treatment guidelines. Methods We identified and reviewed the published data on the use of CPI agents in GTN. Outcome 133 patients were identified who had been treated with CPI for GTN with pembrolizumab (23), avelumab (22), camrelizumab (57), toripalimab (15) or other anti-PD-1 agents (16), of whom 118 had high risk disease, relapse or multi drug resistant disease, and 15 low risk disease. Overall 85 patients achieved complete remission, 77 (of 118) with high risk disease and 8 (of 15) with low risk disease. 1 patient with complete remission in the high risk group developed a relapse 22 months after anti-PD-1 treatment had been stopped. Treatment was generally well tolerated across studies. Conclusions and Outlook The majority of high risk patients (77/118) treated with CPI are cured and this is particularly relevant amongst those with chemotherapy resistant disease who otherwise have very limited treatment options. Priorities for future research include determining whether these agents have a role earlier in the disease course, the utility of combination with chemotherapy, and effects on future fertility. Treatment availability remains a concern due to the high price of these agents.

Predictors for single-agent resistance in FIGO score 5 or 6 gestational trophoblastic neoplasia: a multicentre, retrospective, cohort study.

BACKGROUND: Patients with gestational trophoblastic neoplasia who have an International Federation of Gynaecology and Obstetrics (FIGO) risk score of 5 or 6 usually receive non-toxic single-agent chemotherapy as a first-line treatment. Previous studies suggest that only a third of patients have complete remission, with the remaining patients requiring toxic multiagent chemotherapy to attain remission. As stratification factors are unknown, some centres offer multiagent therapy upfront, resulting in overtreatment of many patients. We aimed to identify predictive factors for resistance to single-agent therapy to inform clinicians on which patients presenting with a FIGO score of 5 or 6 are likely to benefit from upfront multiagent chemotherapy. METHODS: We did a multicentre, retrospective, cohort study of patients with gestational trophoblastic neoplasia presenting with a FIGO score of 5 or 6, who received treatment at three gestational trophoblastic neoplasia reference centres in the UK, Brazil, and the USA between Jan 1, 1964, and Dec 31, 2018. All patients who had been followed up for at least 12 months after remission were included. Patients were excluded if they had received a non-standard single-agent treatment (eg, etoposide); had been given a previously established first-line multiagent chemotherapy regimen; or had incomplete data for our analyses. Patient data were retrieved from medical records. The primary outcome was the incidence of chemoresistance after first-line or second-line single-agent chemotherapy. Variables associated with chemoresistance to single-agent therapies were identified by logistic regression analysis. In patient subgroups defined by choriocarcinoma histology and metastatic disease status, we did bootstrap modelling to define thresholds of pretreatment human chorionic gonadotropin concentrations and identify groups of patients with a greater than 80% risk (ie, a positive predictive value [PPV] of 0·8) of resistance to single-agent chemotherapy. FINDINGS: Of 5025 patients with low-risk gestational trophoblastic neoplasia, we identified 431 patients with gestational trophoblastic neoplasia presenting with a FIGO risk score of 5 or 6. All patients were followed up for a minimum of 2 years. 141 (40%) of 351 patients developed resistance to single-agent treatments and required multiagent chemotherapy to achieve remission. Univariable and multivariable logistic regression revealed metastatic disease status (multivariable logistic regression analysis, odds ratio [OR] 1·9 [95% CI 1·1-3·2], p=0·018), choriocarcinoma histology (3·7 [1·9-7·4], p=0·0002), and pretreatment human chorionic gonadotropin concentration (2·8 [1·9-4·1], p<0·0001) as significant predictors of resistance to single-agent therapies. In patients with no metastatic disease and without choriocarcinoma, a pretreatment human chorionic gonadotropin concentration of 411 000 IU/L or higher yielded a PPV of 0·8, whereas in patients with either metastases or choriocarcinoma, a pretreatment human chorionic gonadotropin concentration of 149 000 IU/L or higher yielded the same PPV for resistance to single-agent therapy. INTERPRETATION: Approximately 60% of women with gestational trophoblastic neoplasia presenting with a FIGO risk score of 5 or 6 achieve remission with single-agent therapy; almost all remaining patients have complete remission with subsequent multiagent chemotherapy. Primary multiagent chemotherapy should only be given to patients with metastatic disease and choriocarcinoma, regardless of pretreatment human chorionic gonadotropin concentration, or to those defined by our new predictors. FUNDING: None. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Following chemotherapy for gestational trophoblastic neoplasia, do residual lung lesions increase the risk of relapse?

BACKGROUND: There remains uncertainty about the prognostic significance of residual lung lesion on imaging after completion of treatment of low- or high-risk gestational trophoblastic neoplasia (GTN). Here, we determine if such residual lung lesions are associated with an increased risk of relapse. METHODS: We retrospectively screened our electronic database to identify patients with low- or high-risk GTN and lung metastases between 2004 and 18. Recurrences among patients with or without residual lung lesions on imaging were compared. Chi square analysis and Kaplan-Meier survival curves were constructed. As the numbers of cases were low, we combined this data with our previously published and non-overlapping patient cohort (1995-2004). RESULTS: Of 1304 GTN patients treated at our centre between 2004 and 18, 99 had lung metastases without other distant sites. There were 40 patients (40.4%) with residual lung lesions. Whilst an increased rate of relapse was observed among patients with residual lung lesions (4/40; 10.0%) compared to without such lesions (3/59; 5.1%), this difference was not statistically significant (p = .35). By combining the data with our previous cohort, there was an increase in relapse rate of patients with residual lung lesions (5/63; 7.9%) compared to those without such lesions (4/112; 3.6%). However, this difference was also not statistically significant (p = .21). CONCLUSION: Residual lung lesions on imaging after completion of GTN treatment are common. However, this finding did not statistically increase relapse rate. Due to low number of recurrent events, a multi-centre, larger dataset would be needed to provide more definitive evidence.

When to stop human chorionic gonadotrophin (hCG) surveillance after treatment with chemotherapy for gestational trophoblastic neoplasia (GTN): A national analysis on over 4,000 patients.

OBJECTIVE: To determine the optimal duration of human chorionic gonadotrophin (hCG) surveillance following treatment for low and high risk gestational trophoblastic neoplasia (GTN) and establish whether the current surveillance protocol that recommends life-long hCG monitoring requires revision. METHODS: A population-based cohort study was undertaken using a national registry, comprising patients from both tertiary trophoblastic disease treatment units in the UK (London and Sheffield). All patients who received chemotherapy for low or high risk GTN in the UK between 1958 and 2014 in London and 1973 and 2015 in Sheffield (n = 4201) were included in the study. Patients with placental site trophoblastic tumours and epithelioid trophoblastic tumours were excluded due to their distinct clinical behavior, treatment and follow-up requirements. The risk of recurrence with time following completion of chemotherapy for low or high risk GTN was measured. RESULTS: The overall risk of relapse in this national cohort of 4201 patients was 4.7% (198/4201) with a median time to recurrence of 117.5 days (range 9 days to 6.54 years). The greatest risk of recurrence occurred in the first year after completing treatment for either low or high risk GTN measuring 72.7% (n = 112) or 86.4% (n = 38), respectively. The subsequent recurrence risk reduced over time with none observed beyond 7 years. CONCLUSIONS: The absence of any recurrences beyond seven years following completion of chemotherapy for GTN indicates that the UK policy of life-long hCG surveillance is unnecessary. Our revised conservative protocol recommends stopping after 10 years.

Anti-Müllerian Hormone in Patients Treated with Chemotherapy for Gestational Trophoblastic Neoplasia Does Not Predict Short-Term Fertility.

OBJECTIVE: Serum anti-Müllerian hormone (AMH) is an emerging indicator of ovarian reserve which may be predictive of reproductive capacity. Although AMH levels decline with chemotherapy, little is known about the relevance of this to subsequent fertility, and we set out to evaluate this association in patients with gestational trophoblastic neoplasia (GTN). STUDY DESIGN: The GTN database of our national referral center was screened from 2008-2012 for patients undergoing AMH testing, and subsequent fertility outcomes were reviewed. RESULTS: Of 470 treated patients, 3 underwent AMH testing for evaluation of potential subfertility 4-13 months following multiagent chemotherapy, with levels rangingfrom 0.07-4.62 pmol/L. All 3 were counseled by independent fertility specialists of the low probability of subsequent conception but went on to initiate spontaneously conceived pregnancies within 2-9 months, resulting in healthy infants. CONCLUSION: Low serum AMH is not a reliable predictor of reduced short-term fertility postchemotherapy for GTN and should be interpreted with caution when counseling patients in this setting.

Safety and Efficacy of Modified FOLFIRINOX for Advanced Pancreatic Adenocarcinoma: A UK Single-Centre Experience.

OBJECTIVES: The combination of 5-fluorouracil (5-FU), irinotecan and oxaliplatin (FOLFIRINOX) is considered the first-line chemotherapy for fit patients with advanced pancreatic ductal adenocarcinoma (PDAC) but carries an unfavourable adverse event (AE) profile. We retrospectively evaluated the tolerability and efficacy of a modified FOLFIRINOX (mFOLFIRINOX) regimen: intravenous oxaliplatin 85 mg/m2, irinotecan 135 mg/m2, folinic acid 400 mg/m2 and 5-FU infusion 2,400 mg/m2 over 46 h, with routine subcutaneous filgrastim on a 14-day cycle. METHODS: Records of 18 patients with advanced PDAC who received treatment with mFOLFIRINOX were reviewed. Imaging of measurable disease was assessed for response, and survival was measured from the date of commencing chemotherapy to disease progression and/or death. RESULTS: Grade 3 or 4 AEs (n; %) included vomiting (5; 28), nausea (4; 22), diarrhoea (3; 17) and non-neutropaenic fever (3; 17). For patients with stage IV disease, 12/15 (80%) achieved at least stable disease as the best radiological response, with 7/15 (47%) objective responses. In this subgroup, median overall and progression-free survival were 9.3 months (95% CI 8.3-10.4) and 7.2 months (95% CI 4.7-9.6), respectively. CONCLUSION: Compared to full-dose FOLFIRINOX, our modified regimen resulted in lower haematological but only marginally improved non-haematological toxicity rates, with comparable efficacy outcomes. Prospective studies are required to validate these findings.

Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function.

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

What is the optimal duration, dose and frequency for anti-PD1 therapy of non-small cell lung cancer?

Over the past decade, immune checkpoint inhibitors (ICIs) have transformed the management of multiple malignancies including lung cancer. However, the optimal use of these agents in terms of duration, dose and administration frequency remains unknown. Focusing on anti-PD1 agents nivolumab and pembrolizumab in the context of non-small cell lung cancer, we argue that several lines of evidence suggest current administration regimens of these drugs may result in overtreatment with potentially important implications for cost, quality of life and toxicity. This review summarizes evidence for the scope to optimize anti-PD1 regimens, the limitations of existing data and potential approaches to solve these problems including with a novel multi-arm clinical trial design implemented in the recently opened REFINE-Lung study.

REFINE (REduced Frequency ImmuNE checkpoint inhibition in cancers): A multi-arm phase II basket trial testing reduced intensity immunotherapy across different cancers.

BACKGROUND: Immune checkpoint inhibitors (ICI) have revolutionised treating advanced cancers. ICI are administered intravenously every 2-6 weeks for up to 2 years, until cancer progression/unacceptable toxicity. Physiological efficacy is observed at lower doses than those used as standard of care (SOC). Pharmacodynamic studies indicate sustained target occupancy, despite a pharmacological half-life of 2-3 weeks. Reducing frequency of administration may be possible without compromising outcomes. The REFINE trial aims to limit individual patient exposure to ICI whilst maintaining efficacy, with potential benefits in quality of life and reduced drug treatment/attendance costs. METHODS/DESIGN: REFINE is a randomised phase II, multi-arm, multi-stage (MAMS) adaptive basket trial investigating extended interval administration of ICIs. Eligible patients are those responding to conventionally dosed ICI at 12 weeks. In stage I, patients (n = 160 per tumour-specific cohort) will be randomly allocated (1:1) to receive maintenance ICI at SOC vs extended dose interval. REFINE is currently recruiting UK patients with locally advanced or metastatic renal cell carcinoma (RCC) who have tolerated and responded to initial nivolumab/ipilimumab, randomised to receive maintenance nivolumab SOC (480 mg 4 weekly) vs extended interval (480 mg 8 weekly). Additional tumour cohorts are planned. Subject to satisfactory outcomes (progression-free survival) stage II will investigate up to 5 different treatment intervals. Secondary outcome measures include overall survival, quality-of-life, treatment-related toxicity, mean incremental pathway costs and quality-adjusted life-years per patient. REFINE is funded by the Jon Moulton Charity Trust and Medical Research Council, sponsored by University College London (UCL), and coordinated by the MRC CTU at UCL. Trial Registration ISRCTN79455488. NCT04913025 EUDRACT #: 2021-002060-47. CTA 31330/0008/001-0001; MREC approval: 21/LO/0593. REFINE Protocol version 4.0.

Platinum-based chemotherapy attenuates the effector response of CD8 T cells to concomitant PD-1 blockade.

PURPOSE: Combination of chemotherapy (CT) with programmed cell death (PD)-1 blockade is a front-line treatment for lung cancer. However, it remains unknown whether and how CT affects the response of exhausted CD8 T cells to PD-1 blockade. EXPERIMENTAL DESIGN: We used the well-established mouse model of T cell exhaustion with chronic lymphocytic choriomeningitis virus (LCMV) infection to assess the effect of CT (cisplatin+pemetrexed) on T cell response to PD-1 blockade, in the absence of the impact of CT on antigen release and presentation observed in tumor models. RESULTS: When concomitantly administered with PD-1 blockade, CT affected the differentiation path of LCMV-specific CD8 T cells from stem-like to transitory effector cells, thereby reducing their expansion and production of interferon (IFN)-γ. After combination treatment, these restrained effector responses resulted in impaired viral control, compared to PD-1 blockade alone. The sequential combination strategy, where PD-1 blockade followed CT, proved to be superior to the concomitant combination, preserving the proliferative response of exhausted CD8 T cells to PD-1 blockade. Our findings suggest that the stem-like CD8 T cells themselves are relatively unaffected by CT partly because they are quiescent and maintained by slow self-renewal at the steady state. However, upon the proliferative burst mediated by PD-1 blockade, the accelerated differentiation and self-renewal of stem-like cells may be curbed by concomitant CT, ultimately resulting in impaired overall CD8 T cell effector functions. CONCLUSIONS: In a translational context, we provide a proof-of-concept to consider optimizing the timing of chemo-immunotherapy strategies for improved CD8 T cell functions.

Differential cytopathology and kinetics of measles oncolysis in two primary B-cell malignancies provides mechanistic insights.

Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

Sensitive screening of single nucleotide polymorphisms in cell free DNA for diagnosis of gestational tumours.

Tumours expressing human chorionic gonadotropin (hCG), the majority of which are difficult to biopsy due to their vascularity, have disparate prognoses depending on their origin. As optimal management relies on accurate diagnosis, we aimed to develop a sensitive cell free DNA (cfDNA) assay to non-invasively distinguish between cases of gestational and non-gestational origin. Deep error-corrected Illumina sequencing of 195 common single nucleotide polymorphisms (SNPs) in cfDNA and matched genomic DNA from 36 patients with hCG-secreting tumours (serum hCG 5 to 3,042,881 IU/L) and 7 controls with normal hCG levels (≤4 IU/L) was performed. cfDNA from confirmed gestational tumours with hCG levels ranging from 1497 to 700,855 IU/L had multiple (n ≥ 12) 'non-host' alleles (i.e. alleles of paternal origin). In such cases the non-host fraction of cfDNA ranged from 0.3 to 40.4% and correlated with serum hCG levels. At lower hCG levels the ability to detect non-host cfDNA was variable, with the detection limit dependent on the type of causative pregnancy. Patients with non-gestational tumours were identifiable by the absence of non-host cfDNA, with copy number alterations detectable in the majority of cases. Following validation in a larger cohort, our sensitive assay will enable clinicians to better inform patients, for whom biopsy is inappropriate, of their prognosis and provide optimum management.