Abstract Investigating the impact of immune-modulating therapies on mRNA vaccine efficacy transcends the immediate context of the COVID-19 pandemic. This study focuses on the differential immune responses to the third dose of COVID-19 mRNA vaccine among healthy volunteers, cancer patients treated with immune-checkpoint inhibitors (ICIs), and those treated with the anti-CD20 antibody rituximab. Utilizing RNA sequencing, serology, and interferon-γ release assessment, we charted the temporal dynamics of the immune response in such cohorts. Our findings indicate that ICIs maintain an immune profile similar to that of healthy individuals, whereas treatment with rituximab is associated with impairment of type I interferon response and the upregulation of transcripts pertaining to regulatory T cells, with a global dysfunction of both humoral and cellular immunity. This research deepens our understanding of the sophisticated interplay within the immune system in health and disease states, potentially informing therapeutic strategies across a spectrum of immunological conditions. Significance statement Our study examines how cancer treatments that modify the immune system affect transcriptional, serological, and cellular responses to a model for repeated antigenic stimulation in humans, represented by the SARS-CoV-2 booster vaccine. Specifically, we investigated patients treated with rituximab (RTX), which impairs antibody production, and immune checkpoint inhibitors (ICI), which can trigger autoimmune disorders. We discovered that RTX-treated patients not only exhibit a reduced antibody response but actually show a diminished interferon-mediated immune response, indicating a broader immune disruption than anticipated. Conversely, ICI-treated patients responded to the vaccine similarly to healthy individuals, suggesting that fears of adverse vaccine reactions in these patients may be unfounded. This research highlights important considerations for the clinical management of cancer patients receiving these treatments.
Vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic (VEXAS) syndrome, first identified by Beck et al.,1 is an acquired monogenic autoinflammatory disorder characterized by multisystemic relapsing inflammation, often accompanied by multilineage cytopenia, venous thromboembolism, myelodysplastic syndrome and monoclonal gammopathy. VEXAS is caused by post-zygotic mutations of the ubiquitin-like modifier activating enzyme 1 (UBA1) gene within haematopoietic stem cells, resulting in the disruption of the ubiquitin–proteasome pathway.1, 2 Recent research indicates that these aberrations ultimately cause abnormal myeloid cell cycling and increased cytokine production by monocytes.3, 4 Nevertheless, the pathophysiology of VEXAS remains only partially understood, preventing the identification of actionable therapeutic targets. Consistently with these knowledge gaps, few therapeutic options are currently available for the management of VEXAS. Indeed, while high-dose steroids are beneficial during disease flares, their long-term utilization is discouraged by their side effects. On the other hand, conventional disease-modifying anti-rheumatic drugs are scarcely useful for the management of inflammatory symptoms.2, 5, 6 Recent retrospective studies have identified Janus kinase inhibitors (JAKi) as effective therapeutic agents for patients with VEXAS.6 Of these, ruxolitinib, a dual inhibitor of JAK1 and JAK2, is associated with higher response rates compared to other JAKi.6 However, the mechanisms by which patients with VEXAS respond more to ruxolitinib than other JAKi are still unclear. Addressing this knowledge gap is critical for discovering the specific actionable targets in VEXAS, possibly allowing for the optimization of current therapeutic strategies. To dissect the molecular characteristics of response to dual JAK inhibition, we charted the transcriptomic response to therapy in one patient with VEXAS treated with ruxolitinib. The patient, a 63-year-old Caucasian man, was diagnosed with VEXAS by genetic testing in April 2023 following an extensive diagnostic work-up for unexplained inflammatory symptoms, empirically treated with corticosteroids. VEXAS coexisted with WHO-defined myelodysplastic syndrome with low blasts7 and immunoglobulin M monoclonal gammopathy of undetermined significance (MGUS). Azacytidine was initiated in mid-May 2023, with contemporary steroid tapering. By early June, the patient required hospitalization due to high remittent fever, diffuse purpuric rash and enanthem. The biopsy of a purpuric skin lesion revealed dermic neutrophil and lympho-monocytic infiltrates positive for myeloperoxidase, confirming the diagnosis of VEXAS flare. Following initial treatment with intravenous steroids, ruxolitinib was initiated as steroid-sparing agent, resulting in complete clinical remission within 2 weeks (Figure 1A). Ruxolitinib was later discontinued due to worsening pancytopenia, necessitating red cell transfusions. Azacytidine was then resumed, showing good tolerance and a sustained response. To comprehensively profile with high temporal resolution of the response to ruxolitinib, we performed serial blood RNA-sequencing (RNA-seq) coupled with bulk and single-cell RNA-seq (scRNA-seq) of VEXAS skin lesions (Figure 1A). Peripheral blood samples were obtained at multiple time points during treatment with ruxolitinib throughout patient's hospitalization and discharge. Specifically, blood samples were collected on day 0 before ruxolitinib initiation and on days 1, 3, 5, 6, and 11 after treatment started. Formalin-fixed, paraffin-embedded skin biopsies obtained per clinical practice were collected before starting steroid therapy (2 days before ruxolitinib initiation, day −2) and on day 5 after ruxolitinib initiation for bulk RNA-seq. Additionally, a fresh skin tissue sample was collected on day 5 for scRNA-seq. A detailed description of the materials and methods, along with the ethics statement, is provided in the Supporting Information. To verify whether blood RNA-seq was informative of disease activity, we first assessed a transcriptomic VEXAS-specific signature identified by Kosmider et al. and comprising IL-1α, IL-1β, IL-18, TGF-α, IL-7, LGALS3, S100A8 and S100A9.4 Treatment with ruxolitinib was associated with a prompt downregulation of this signature (Figure 1B), in parallel with the progressive resolution of symptoms. Indeed, the VEXAS signature positively correlated with several laboratory markers of inflammatory activity assessed per clinical practice, including red cell distribution width, neutrophil percentage and absolute count, C-reactive protein, ferritin and lactate dehydrogenase. Conversely, it was negatively associated with monocyte percentage and absolute count, mean corpuscular haemoglobin concentration, as well as lymphocyte percentage and absolute count (Figure 1C; Table S1). To shed light on the molecular determinants of response to JAK inhibition, we next profiled the transcriptional activity of the JAK–signal transducer and activator of transcription proteins (STAT) signalling pathway, observing a progressive downregulation of the JAK–STAT signature following ruxolitinib initiation (Figure S1). Notably, when considering the individual transcripts of JAK1 and JAK2, we observed a marked downregulation of JAK2, while JAK1 expression did not change over time (Figure 1C). JAK2 was positively associated with the VEXAS signature, as well as with interferon signalling, a key inflammatory pathway downstream of JAK-STAT. JAK2 expression was also associated with plasma cell response, indicative of MGUS activity. JAK1 displayed an opposite trend compared to JAK2 (Figure 1D). Considering bulk RNA-seq performed on VEXAS skin lesions, the transcriptomic changes occurring after treatment initiation recapitulated those observed in blood, with the downregulation of VEXAS and interferon signatures, coupled with JAK2, but not JAK1, transcript decrease (Figure 2A). Immune cell deconvolution revealed a concomitant shift from a pro-inflammatory environment to a tissue repair status, with the complete loss of neutrophils and monocytes and a switch of macrophages from an M1 pro-inflammatory phenotype towards an M2 anti-inflammatory one (Figure 2B), consistent with clinical improvement. scRNA-seq analysis performed on fresh skin tissue collected on day 5 further confirmed the differential expression of JAK1 and JAK2 genes after treatment initiation (Figure 2C). Notably, both genes were mainly expressed on clusters of fibroblasts, vascular endothelial cells and pericytes. Indeed, JAK-STAT signalling is known to modulate endothelial permeability, inducing a pro-adhesive and pro-coagulant switch in inflammatory conditions.8 Overall, our results indicate that JAK2 is a critical actionable target in patients with VEXAS, which has been suggested in clinical reports but never described from a molecular standpoint.6 Despite the efficacy of ruxolitinib, its discontinuation due to pancytopenia in the above-described case underscores the need for careful management of its haematological side effects. Our findings provide a strong pathophysiological basis to investigate the efficacy of selective anti-JAK2 molecules in patients with VEXAS, potentially reducing the rate of therapy-related side effects while preserving the efficacy in inducing and maintaining clinical remission. Conceptualization: GZ, LF and AlB, methodology: GZ, LF and MD, data collection: NG, AnB, BC, MS, AC, MD, IL, TV, GR, MD, IS and AG, data analysis: LF, GZ, FR, MD, MC and AC, software: LF, formal analysis: AC, MD, FR, LF, AlB and GZ, writing and review: all authors, visualization: LF, GZ and FR, supervision: AlB, LF and GZ, project administration: MD and GZ and funding acquisition: AlB and GZ. This work was supported by Associazione Italiana Linfoma, Leucemie e Mieloma Multiplo (AIL to AlB), Alleanza Contro il Cancro (ACC), Fondazione AIRC – Associazione Italiana per la Ricerca contro il Cancro (IG21761 to GZ), Research Projects of National Interest (Italian Ministry of University and Research, ID: M4.C2.1.1 to GZ), CURIOSITY driven research funds (University of Genoa to GZ) and unrestricted donations from the Benvenuto and Marra families. GZ wishes to thank Dr. P. Blandini for his ever-insightful criticisms of the technical aspects of the present work. GZ declares consultancy fees from Menarini Stemline and holds the co-ownership of Immunomica Ltd. The remaining authors have no conflict of interest to disclose. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Little is known about SARS-CoV-2 seroconversion in asymptomatic patients affected by solid cancer, and whether it is associated with specific transcriptomics changes in peripheral blood mononuclear cells (PBMC).Patients affected by solid cancer treated in a top comprehensive cancer center in Italy during the first COVID-19 pandemic wave, and negative for COVID-19-symptoms since the first detection of COVID-19 in Italy, were prospectively evaluated by SARS-CoV-2 serology in the period between April 14th and June 23rd 2020. Follow-up serologies were performed, every 21-28 days, until August 23rd 2020. All SARS-CoV-2 IgM + patients underwent confirmatory nasopharyngeal swab (NPS). PBMCs from a subset of SARS-CoV-2 IgM + patients were collected at baseline, at 2 months, and at 7 months for transcriptome sequencing.SARS-CoV-2 serology was performed on 446 of the 466 recruited patients. A total of 14 patients (3.14%) tested positive for at least one SARS-CoV-2 immunoglobulin in the period between April 14th and August 23rd 2020. Incidence of SARS-CoV-2 IgM decreased from 1.48% in the first month of the accrual to 0% in the last month. Viral RNA could not be detected in any of the NPS. PBMC serial transcriptomic analysis showed progressive downregulation of interleukin 6 upregulated signatures, chemokine-mediated signaling and chemokine-chemokine receptor KEGG pathways. B- and T-cell receptor pathways (p-values = 0.0002 and 0.017 respectively) were progressively upregulated.SARS-CoV-2 seroconversion rate in asymptomatic patients affected by solid cancer is consistent with that of asymptomatic COVID-19 assessed in the general population through NPS at the peak of the first wave. Transcriptomic features over time in IgM + asymptomatic cases are suggestive of previous viral exposure.
Background: Androgen receptor signaling inhibitors (ARSis) abiraterone acetate (AA) plus prednisone and enzalutamide (Enza), are currently the most administered first-line treatments for metastatic castration-resistant prostate cancer (mCRPC). AA and Enza have shown similar overall survival (OS) benefits and there is no consensus upon the best option for mCRPC first-line treatment. Volume of disease may represent a useful biomarker to predict response to therapy in such patients. Objectives: In this study, we seek to evaluate the impact of volume of disease on patients treated with first-line AA versus Enza for mCRPC. Design and methods: We retrospectively evaluated a cohort of consecutive patients with mCRPC categorized by volume of disease [high volume (HV) or low volume (LV) per E3805 criteria] at ARSi onset and treatment type (AA or Enza), assessing OS and radiographic progression-free survival (rPFS), from therapy start, as co-primary endpoints. Results: Of the 420 patients selected, 170 (40.5%) had LV and received AA (LV/AA), 76 (18.1%) LV and had Enza (LV/Enza), 124 (29.5%) HV and were given AA (HV/AA), and 50 (11.9%) HV and received Enza (HV/Enza). Among patients with LV, OS was significantly longer when treated with Enza [57.2 months; 95% confidence interval (CI): 52.1–62.2 months] versus AA (51.6 months; 95% CI, 42.6–60.6 months; p = 0.003). Consistently, those with LV receiving Enza showed increased rPFS (40.3 months; 95 CI, 25.0–55.7 months) than those having AA (22.0 months; 95% CI, 18.1–26.0 months; p = 0.004). No significant difference in OS or rPFS was observed in those with HV treated with AA versus Enza ( p = 0.51 and p = 0.73, respectively). In multivariate analysis of patients with LV, treatment with Enza was independently associated with better prognosis than AA. Conclusion: Within the intrinsic limitations of a retrospective design and small population, our report suggests that volume of disease could be a useful predictive biomarker for patients starting first-line ARSi for mCRPC.
Abstract The genomic, epigenetic and metabolic determinants of prostate cancer pathobiology have been extensively studied in epithelial cancer cells. However, malignant cells constantly interact with the surrounding environment—the so-called tumour microenvironment (TME)—which may influence tumour cells to proliferate and invade or to starve and die. In that regard, stromal cells—including fibroblasts, smooth muscle cells and vasculature-associated cells—constitute an essential fraction of the prostate cancer TME. However, they have been largely overlooked compared to other cell types (i.e. immune cells). Indeed, their importance in prostate physiology starts at organogenesis, as the soon-to-be prostate stroma determines embryonal epithelial cells to commit toward prostatic differentiation. Later in life, the appearance of a reactive stroma is linked to the malignant transformation of epithelial cells and cancer progression. In this Review, we discuss the main mesenchymal cell populations of the prostate stroma, highlighting their dynamic role in the transition of the healthy prostate epithelium to cancer. A thorough understanding of those populations, their phenotypes and their transcriptional programs may improve our understanding of prostate cancer pathobiology and may help to exploit prostate stroma as a biomarker of patient stratification and as a therapeutic target.
Non-invasive early diagnosis of cancer is a highly desirable goal in oncology. Plasma proteins have potential application in clinical practice as cancer biomarker. However, their profiling in presence of small tumors, entails technical and biological challenges. Cutting-edge technologies could overcome these limitations. In the present study, we describe preliminary data on the assessment of circulating proteins in early breast cancer (eBC) patients from the RENOVATE trial by employing two high-throughput proteomics approaches. The RENOVATE trial (NCT04781062) enrolled women with suspect breast lesions ≤ 2 cm. They were asked to donate blood sample (detailed information in Ravera et al., BMJ, 2021). Plasma samples from 31 cancer, 29 benign and 20 healthy donors, matched by age, body mass index (BMI), menopausal and smoking status, were tested by aptamer-based proteomics arrays (SomaScan Somalogic®). Nearly 370 proteins were reassessed on the same biospecimens, leveraging an antibody-DNA linker technology assay (Oncology panel, Olink®). Variance analysis was performed using ANOVA. Statistical tests with a p-value < 0.01 were considered significant. Proteomics signatures were identified by a penalized linear model (glmnet R package). The two assays correlation was evaluated using Intra-class correlation (ICC). Out of 7596 proteins, 29 showed a significant differences across the 3 groups. The detected proteins are mainly involved in differentiation, migration, and cell death pathways. The two platforms shared 300 targets; 99% (n=296) passed the quality control step. The two used methodologies displayed moderately consistent results with 22% of probes (n=66) with an ICC index ≥ 0.5. In the present study, we performed a broad analysis of human circulating proteins. We detected proteins associated with early BC related to pathways influencing cancer development. Data were validated by an orthogonal technology. Despite the potentiality in early diagnosis, the application of advanced methodologies is constrained by costs, and the majority of detected biomarkers have not yet been validated by standardized routine assays. We are currently working on the validation of our findings by implementing low-throughput and low-cost methods.
Liquid biopsy has the potential to drastically change clinical practice, paving the way to a novel non-invasive approach for cancer diagnosis and treatment. One of the limitations for the implementation of liquid biopsy in clinical practice is the lack of shared and reproducible standard operating procedures (SOPs) for sample collection, processing and storage. Here, we present a critical review of the literature focusing on the available SOPs to guide liquid biopsy management in research settings and describe SOPs that our laboratory developed and employed in the context of a prospective clinical-translational trial (RENOVATE, NCT04781062). The main aim of this manuscript is to address common issues, towards the implementation of interlaboratory shared protocols for optimized preanalytical handling of blood and urine samples. To our knowledge, this work is one of the few up-to-date, freely available comprehensive reports on trial-level procedures for the handling of liquid biopsy.
