key: cord-0903622-gnfnigjk
authors: Sacconi, Andrea; De Vitis, Claudia; de Latouliere, Luisa; di Martino, Simona; De Nicola, Francesca; Goeman, Frauke; Mottini, Carla; Paolini, Francesca; D’Ascanio, Michela; Ricci, Alberto; Tafuri, Agostino; Marchetti, Paolo; Di Napoli, Arianna; De Biase, Luciano; Negro, Andrea; Napoli, Christian; Anibaldi, Paolo; Salvati, Valentina; Duffy, Darragh; Terrier, Benjamin; Fanciulli, Maurizio; Capalbo, Carlo; Sciacchitano, Salvatore; Blandino, Giovanni; Piaggio, Giulia; Mancini, Rita; Ciliberto, Gennaro
title: Multi-omic approach identifies a transcriptional network coupling innate immune response to proliferation in the blood of COVID-19 cancer patients
date: 2021-10-29
journal: Cell Death Dis
DOI: 10.1038/s41419-021-04299-y
sha: 560079ff90c5444acb2f3685ececefbc13a54e54
doc_id: 903622
cord_uid: gnfnigjk

Clinical outcomes of COVID-19 patients are worsened by the presence of co-morbidities, especially cancer leading to elevated mortality rates. SARS-CoV-2 infection is known to alter immune system homeostasis. Whether cancer patients developing COVID-19 present alterations of immune functions which might contribute to worse outcomes have so far been poorly investigated. We conducted a multi-omic analysis of immunological parameters in peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with and without cancer. Healthy donors and SARS-CoV-2-negative cancer patients were also included as controls. At the infection peak, cytokine multiplex analysis of blood samples, cytometry by time of flight (CyTOF) cell population analyses, and Nanostring gene expression using Pancancer array on PBMCs were performed. We found that eight pro-inflammatory factors (IL-6, IL-8, IL-13, IL-1ra, MIP-1a, IP-10) out of 27 analyzed serum cytokines were modulated in COVID-19 patients irrespective of cancer status. Diverse subpopulations of T lymphocytes such as CD8(+)T, CD4(+)T central memory, Mucosal-associated invariant T (MAIT), natural killer (NK), and γδ T cells were reduced, while B plasmablasts were expanded in COVID-19 cancer patients. Our findings illustrate a repertoire of aberrant alterations of gene expression in circulating immune cells of COVID-19 cancer patients. A 19-gene expression signature of PBMCs is able to discriminate COVID-19 patients with and without solid cancers. Gene set enrichment analysis highlights an increased gene expression linked to Interferon α, γ, α/β response and signaling which paired with aberrant cell cycle regulation in cancer patients. Ten out of the 19 genes, validated in a real-world consecutive cohort, were specific of COVID-19 cancer patients independently from different cancer types and stages of the diseases, and useful to stratify patients in a COVID-19 disease severity-manner. We also unveil a transcriptional network involving gene regulators of both inflammation response and proliferation in PBMCs of COVID-19 cancer patients.

The impact of the COVID-19 pandemic on cancer patients has become a major focus of the investigation. The association of COVID-19 and cancer is responsible for a more severe clinical course with worse outcomes and a lethality rate of up to 25% in these patients [1, 2] , and the risk of adverse outcomes of SARS-CoV-2 infection is significantly higher for patients with cancer versus those without. The development of cancer itself, surgical treatment and related procedures, radiotherapy, and/or systemic treatments may lead to immune suppression [3] , thus increasing the risk of SARS-Cov-2 infection [4] [5] [6] . The negative impact of COVID-19 has been seen across a broad spectrum of cancers [7] [8] [9] [10] . Although cancer by itself may be a risk factor for the infection and evolution of the COVID-19 disease, the biological mechanisms underlying this synergy have been scarcely investigated.

During SARS-CoV-2 infection, a derailed immune response of the host is the main cause of tissue damage and the resulting severity of the disease. Serum cytokines are aberrantly produced in these patients and circulating subsets of immune cells are altered compared to healthy donors. In agreement, lymphocytes' gene expression profiles are deeply modified in Covid-19 patients and these alterations are further enhanced with disease severity [11] [12] [13] . Being lymphopenia, the strongest predictor for severity of disease in COVID-19 patients [14] . An impaired interferon-I (IFN-1) response that leads to a low expression of interferon-stimulated genes coupled with high levels of cytokines represents a hallmark of COVID-19 infection [12, [15] [16] [17] . Few data are available about the modulation of immune cells in COVID-19 patients with cancer. A multicentre study reported a significant decrease in the number of T and NK cells in COVID-19 patients with hematological malignancies when compared to COVID-19 patients [18] . Given the key role of the immune response in both COVID-19 and cancer, we hypothesized that the presence of SARS-CoV-2 in a host immunologically compromised by cancer, could result in specific features useful as prognostic markers of disease progression. Based on this hypothesis, our main purpose was to find immunological features specific to cancer patients infected with SARS-CoV-2 and to unveil how COVID-19 disease impacts the immune system of these patients. To this purpose, using a multiomic experimental approach, we investigated COVID-19 patients affected by neoplasms, serum cytokines, changes in the proportion of circulating subsets of immune cells, and PBMC immunological gene expression profiles. Of note, we identified a gene expression signature, common to different cancer types and independent from stages of the diseases. Moreover, we identified for the first time a network including transcription factors (TFs) known to be relevant for the innate response upon viral infection and cancer chronic inflammation, such as IRF, STAT, and BATF and a master regulator of proliferation, E2F, which underlies aberrant immune and proliferation responses of PBMCs in COVID-19 cancer patients.

We collected blood samples to perform single-cell and molecular immunological analysis by fCyTOF and NanoString technologies of ten COVID-19 patients and four healthy donors (HD). In five out of ten patients, COVID-19 was associated with cancer. We have also included, in the Nanostring analysis, a cohort of ten patients with cancer without COVID-19. The Nanostring results have been further validated by RT-PCR including additional two cancer patients without COVID-19 and 16 cancer COVID-19 patients ( Supplementary Fig. 1 ). Clinical and epidemiological characteristics of this group of patients are reported in Supplementary Table I. According to the criteria reported by the Chinese National Health Commission [19, 20] , we subdivided COVID-19 patients in five critical, eight severe, six mild, three moderate, and four asymptomatic. Among cancer patients, we collected 13 lung cancer, 6 hematological malignancy, 5 gastrointestinal cancer, 2 breast cancer, 2 clear cell renal cell carcinoma, and 5 other malignancies (Supplementary Fig. 1 and Supplementary Table I) .

Symptomatic COVID-19 instigates hypercytokinemia irrespective of cancer status Overproduction of pro-inflammatory cytokines occurs both in COVID-19 diseases and cancer [21, 22] . Herein, we aimed to assess whether COVID-19 modulates differently cytokine production in infected subjects with or without cancer. With the aid of Bio-Plex Pro Human Cytokine 27-Plex Immunoassay, we assessed concomitantly the expression of 27 cytokines in patient sera. As shown in Fig. 1a , principal component analysis (PCA) based on the quantification of the 27 cytokines clearly discriminated a cohort of mild-to-critical COVID-19 subjects from HD. By focusing on the top statistically ranked cytokines we evidenced a subset of 8 cytokines expressed at higher levels in the COVID-19 subjects than in HD sera ( Fig. 1b-d) . Interestingly, an increased upregulation of IP-10 could be observed in male COVID-19 subjects compared to the non-gender segregated group (Fig. 1e) while the same does not happen in HD ( Supplementary Fig. S2 ). Next, we assessed the presence of these eight cytokines in sera from COVID-19 subjects concomitantly affected by cancer, and no statistically significant differences were evidenced between this group and COVID-19 subjects (Fig. 1d) .

In aggregate, we documented that COVID-19 mild-to-severe patients exhibited cytokine overproduction typical of active viral infection and this occurs independently from the cancer comorbidity.

Subclasses of hematopoietic cell populations are modulated in COVID-19 cancer patients To characterize the immune phenotyping of COVID-19 patients, we carried out CyTOF analysis. The immune cellular subsets identified using a 30 antibody panel are listed in Supplementary  Table II . T cells were reduced in COVID-19 patients compared to HD. This reduction was mainly due to a decrease of mucosalassociated invariant T cells (MAIT), NKT, and γδ T cells. The total amount of CD8 + T cells was almost unchanged, but an imbalance of cell subpopulations consisting of an increase in terminal effectors CD8 + T and a decrease of naive CD8 + T was evidenced ( Fig. 2A) . Among B cells, the minority subclass, plasmablasts, as expected robustly increased while B-memory cells slightly decreased in COVID-19 affected patients compared to HD (Fig.  2B ). Within dendritic cells, myeloid and plasmacytoid subpopulations were clearly reduced in COVID-19 patients compared to HD (Fig. 2C) . A slight reduction of NK cells (Fig. 2D ) and non-classical monocytes ( Fig. 2E ) was observed in COVID-19 patients compared to HD. The majority of these modifications have been already reported in the literature confirming the reliability of our results [12, [23] [24] [25] [26] [27] [28] [29] . To search for specific hematopoietic alterations in COVID-19 patients with cancer, we analyzed their immune profiling and compared it to that of COVID-19 patients without cancer. A further slight reduction of T cells was observed in COVID-19 patients with cancer ( Fig. 2A ). This reduction was mainly due to terminal effectors CD8 + T cells that are back to the levels observed in HD, and MAIT-NKT cells. Regarding B cells, the imbalance observed in COVID-19 patients became more pronounced in cancer patients, being plasmablasts further increased and B-memory decreased (Fig. 2B) . The total amount of dendritic ( Fig. 2C) and NK (Fig. 2D ) cells were comparable between COVID-19 patients with cancer and without cancer. The total amount of monocytes was almost unchanged, whereas the non-classical monocytes subpopulation was rather increased in COVID-19 cancer patients compared to non-cancer patients (Fig. 2E) . A graphical representation of the CyTOF data in our cohorts is shown in the Cen-se plot, reporting that different cancer patients affected by different tumors exhibited peculiar differences in the quantity of the various immune cell subpopulations (Supplementary Fig. S3 ).

Identification of a gene signature associated with COVID-19 patients with cancer To further dissect the molecular features distinguishing COVID-19 patients with cancer from COVID-19 patients without cancer, we performed gene expression profiling of total RNA derived from PBMCs of both patient groups. We used the NanoString PanCancer IO 360 Panel that allows analyzing simultaneously the expression of 750 genes involved in the immune response. First, we identified 236 genes whose expression was modulated A. Sacconi et al. between COVID-19 patients (n = 10) and HD (n = 2). To further validate the robustness of our findings, we accessed the gene expression raw data of Terrier's group who analyzed the immunological transcriptional signature of peripheral white blood cells of 50 COVID-19 patients with different degrees of severity [12] . Out of the 236 genes identified in our analysis, we could match 114 genes that overlap with those analyzed by Terrier's group ( Fig. 3a and Supplementary Table III) . Notably, we found that the group of genes that differentiates COVID-19 patients (n = 50) from HD (n = 18) in the cohort reported in Hadjadj J. et al., A. Sacconi et al. 2020 is also able to discriminate COVID-19 subjects versus HD in our cohort (Fig. 3b ). Of note, in the last analysis, our gene signature is clearly able to cluster COVID-19 patients on the basis of clinical severity, too.

Next, we aimed to identify genes whose expression was selectively modulated between COVID-19 patients with cancer or without cancer. Gene Set Enrichment Analysis (GSEA) carried out by looking at all ranked genes revealed surprisingly that genes belonging to interferon α, γ, and β pathways were upregulated in COVID-19 cancer patients (Fig. 3c , upper panel). Since it has been described that one hallmark of the SARS-CoV-2 infection is the ability of the virus to imbalance the IFN pathways [12, [15] [16] [17] , this result surprisingly suggests that the concomitant presence of COVID-19 and cancer triggers a Fig. 3 NanoString gene expression analysis. a Hierarchical clustering of 114 genes significantly modulated between HD (n = 2) and COV patients (n = 10) in Sant'Andrea Hospital cohort. Patients were sorted for the gravity of disease from healthy to critical status. HD reveals a different gene expression profile from disease-affected patients. b The same panel of genes was validated on a published cohort of 45 samples, including 13 HD and 32 COV patients. Hierarchical clustering showed a very similar expression profile. c Gene Set Enrichment Analysis (GSEA) was conducted considering a ranked list of all 750 genes of the NanoString PanCancer IO 360 Panel. Ranking was based on a score evaluated considering the sign of the modulation and the P value from permutation test between COV/cancer (n = 5) and COV patients (n = 5). Enriched gene set for activated genes and deactivated genes are represented on the left and on the right panel, respectively. Statistical significance is shown by a false discovery rate and represented as a color scale. Dimension of circles indicates the percentage of genes included in the gene set enrichment and all the pathways are sorted by the negative or positive normalized enrichment score (NES). The signature is able to discriminate HD (n = 2) from COV patients (n = 5). A difference is also evident between COV/cancer (n = 5) and COV patients (n = 5).

specific inflammatory response. Intriguingly, GSEA enriched for E2F cell cycle-related targets gene, cell cycle, G2M checkpoint, and DNA repair resulted to be upregulated in COVID-19 cancer patients when compared to those without cancer (Fig. 3c , upper panel).

Hadjadj et al. reported two gene signatures, which were related to type I IFN-related genes and cytokine/chemokines, respectively, and allowed to classify COVID-19 symptomatic patients in three groups: mild-to-moderate, severe, and critical. By applying these signatures to our patient cohort, we found that they were able to discriminate COVID-19 patients from HD (Fig. 4a, b) . Interestingly, unlike the cytokines/chemokines signature, type I IFN-related genes were modulated in COVID-19 cancer patients when compared to COVID-19 patients (Fig. 4c, d) . This was evident in both principal component (PC)1 (53.5%) and PC2 (16.5%) analysis components (Fig. 4c) .

Based on these findings, we looked for a gene expression signature capable to discriminate between COVID-19 patients with cancer or without cancer. Bioinformatics analysis allowed us to identify a signature composed of 19 genes whose expression was statistically significantly modulated in COVID-19 cancer patients (n = 5) with respect to both HD (n = 2) and COVID-19 subjects without cancer (n = 5) (Fig. 5a-d) . Of note, the PCA analysis performed with COVID-19 cancer, COVID-19, and cancer patients clearly separates the three groups from each other and from HDs (Fig. 5b) . A group of six genes was upregulated, whereas 13 were downregulated (Fig. 5a, c, d) .

To further validate the specificity of the genes significantly modulated in COVID-19 cancer patients, we carried out a similar analysis on the RNA from PBMCs of ten cancer patients negative for COVID-19. Notably, we found that 10 out of the 19 genes previously identified were also differentially regulated comparing COVID-19 cancer patients with cancer patients without COVID-19 ( Fig. 6a-c) . The majority of upregulated genes belong to the interferon signaling and cytotoxicity pathways while several downregulated genes fall into antigen presentation functional annotated category (https:// www.nanostring.com/products/ncounter-assays-panels/oncology/ pancancer-io-360/) ( Supplementary Fig. S4) ). To further validate the robustness of the gene signature obtained by Nanostring, we analyzed by real-time PCR the expressions of the ten genes of interest. In total, 18 new patients were included in the analysis with a final validation cohort consisting of 12 cancer and 21 COVID-19 cancer patients (Supplementary Fig. 1 and Supplementary Table I) .

Overall, the entire gene signature was confirmed, being downregulated and upregulated genes differentially expressed in the validation cohorts (Fig. 6d) . Focusing on single genes, five out of ten genes (AXIN1, IFIT1, PTGS2, IFIT3, MX1) are significantly deregulated in the new COVID-19 cancer patient cohort (Fig. 6e) . Interestingly, the validated gene signature is independent of cancer type comparing the gene signature in the lung cancer cohort (being the most representative) versus the other types of cancer ( Supplementary Fig.  S5) . Intriguingly, two genes, PTGS2 and CXCL1, are upregulated in COVID-19 cancer patients in a gender-dependent-manner being upregulated in males but not in females (Supplementary Fig. S6 ).

In aggregate, our findings identify a restricted gene signature that was able to discriminate, in a confirmatory consecutive cohort, COVID-19 cancer patients from both COVID-19 patients without cancer and cancer patients without COVID-19.

Next, we subdivided COVID-19 cancer patients according to COVID-19 disease severity. Notably, the ten gene signature shows a severity-dependent trend for downregulated genes (Fig. 7a) , while the same was not observed for upregulated genes (Fig. 7b) . Focusing on single-gene expression, we observed that two genes LILRA1 and CD1C were significantly downregulated in a disease severity-dependent-manner (Fig. 7c) . CXCL1 was downregulated in asymptomatic subjects while it was upregulated in mild/ moderate, severe, and critical patients (Fig. 7d) . IFIT1, IFIT3, and MX1 were upregulated in all severity degrees and further increased in asymptomatic subjects (Fig. 7e) . These results indicate that a specific PBMC gene signature occurs in asymptomatic COVID-19 cancer patients, suggesting its usefulness in the identification of this status.

An immune and proliferation transcriptional network is activated in COVID-19 cancer patients Taking advantage of the Genome Browser ChIP-seq databases at the University of California Santa Cruz (UCSC) (ENCODE tracks), we looked for a common binding signature of transcription factors to the promoters of the genes modulated in COVID-19 cancer patients. The query to the UCSC genome browser has been restricted to the available cells of blood origin (Supplementary  Table IV ). Since in the GSEA analysis IFN α, γ, and β pathways as well as E2F cell cycle-related targets were the most significantly modulated, we focussed our attention on the IRF and E2F family members (Supplementary Table V) . Up to nine IRF members have been identified [30] . We focused on IRF1-5 for which ChIP-seq data are available and found that IRF and E2F family members bind to 12 and 10 promoters out of 19, respectively. Of note, all upregulated genes in the COVID-19 cancer signature contain a binding site for IRF family members in the promoter region spanning 10.000 kb upstream the start site and the first intron (Fig. 8) . IRF family members bind 5 out of 13 promoters of the downregulated genes (Table 1 and Supplementary Fig. S7 ). Based on the role of STAT family members on the interferon pathways [31] , we also investigated their recruitment onto the 19 promoters. STATs form a family of 7 members while STAT ChIP-seq available data are for STAT1, 2, 3, and 5a (Supplementary Table V) . STAT members are recruited on 14 out 19 promoter regions and, as in the case of IRF, all upregulated genes in the COVID-19 cancer signature contain a binding for STATs (Table 1 and Fig. 8 ). It has been recently shown that either cancer or SARS-CoV-2 infection mediates the activity of the IRF4-BATF pathway [32, 33] activating IRF-mediated transcription. In agreement, among the ten promoters bound by BATFs, eight are also bound by IRFs. On the contrary, the binding of E2F family members occurs on the regulatory regions of three upregulated and eight downregulated genes ( Table 1 , Fig. 8 and Supplementary Fig. S7 ). We also assessed the binding of acetylases (p300) and deacetylases (HDACs) proteins and observed both enzymes mostly bind the same promoter regions in agreement with the knowledge that the proper transcriptional activity of target promoters depends on the balanced activities of the two enzymes [34, 35] (Table 1 , Fig. 8 and Supplementary Fig. S7) . Reassuringly, the analysis of ChIP-seq datasets revealed in vivo direct binding of IRF, STAT, and E2F family members onto the indicated promoters. The concomitant binding of IRF and STAT on all promoters of the upregulated genes and five of the downregulated once indicates a transcriptional immune signature specifically acting on COVID-19 cancer patients. Moreover, the binding of promoters of both up-and downregulated genes suggests that IRFs, STATs, and E2Fs Gene expression signature modulated between COVID-19 patients affected by cancer and COVID-19 patients without cancer. a Volcano plot shows P values and related log2 fold change of genes comparing cancer patients and no-cancer patients, both affected by COVID-19. Statistically significance was evaluated by permutation test setting the threshold at 5%. Significant genes are highlighted. b Principal component analysis of the 19 genes on HDs (n = 2), COV (n = 5), cancer (n = 10), and COV/cancer (n = 5) patient groups. c Unsupervised Hierarchical Clustering of the 19-gene signature found to be significantly modulated between COV/cancer (n = 5) and COV patients (n = 5). The signature seems to be able to clearly separates the two groups of patients. d Box plot of the distributions of the 19-gene signature in HDs (n = 2), COV (n = 5), cancer (n = 10), and COV/cancer (n = 5) patient groups. Six genes were statistically significant upregulated:IFI6 (P = 0. contribute to aberrant gene regulation in COVID-19 cancer patients either activation or repression of transcription. This effect has been already shown for other diseases [31, 36, 37] .

In aggregate, we identify a transcriptional network coupling innate immune response and cell proliferation in COVID-19 cancer patients.

In the present manuscript, we aimed to provide initial insights on the impact of COVID-19 on immune-circulating cells in cancer patients. Unexpectedly we unveiled a common gene signature in patients with different neoplasms and different degrees of COVID-19 disease. Albeit limited in number (COVID-19 n = 12 and COVID/ Cancer n = 21), our COVID/Cancer cohort is to date the largest described where gene expression has been investigated in depth. We are aware that the heterogeneity of our COVID/Cancer cohort may result in the loss of cancer-specific gene signatures, however, we believe that the finding of a gene expression signature common to different cancer types and diseases stages could reveal interesting aspects worth further investigation.

To date, there are still scarce evidence on the molecular basis of the severe adverse effects to which COVID-19 cancer patients undergo. Uncertain results emerge from the comparison of both ACE2 and TMPRSS2 expression and functional activities between COVID-19-positive subjects and cancer patients [38, 39] . The aberrant immune response is a common event for both SARS-CoV-2 infection and cancer. This prompted an unprecedented research focus to identify immune factors that discriminate COVID-19 patients with or without cancer. Herein, we observed that serum production of well-recognized inflammatory mediators of the "hypercytokinemia" typical of COVID-19-positive subjects [40] were not differentially affected by the cancer status of SARS-CoV-2-infected patients. CyTOF cell subpopulation immune-profile revealed a small reduction of T cells in COVID-19 patients, further evidenced in COVID-19 cancer patients. This reduction was mainly due to terminal effectors CD8 + T cells whose amount in COVID-19 cancer patients became similar to that of HD, and MAIT-NKT cells [41, 42] . A strong reduction of T cells has been described in severe/critical COVID-19 patients [43] , while we observe a small reduction of these cells. One explanation should reside in the clinical severity heterogeneity in our COVID-19 cohort. Regarding B cells, the imbalance observed in COVID-19 patients became worse in COVID-19 cancer patients, being plasmablasts further increased and B memory decreased.

Notably, we identified a five-gene signature that was modulated between COVID-19, cancer, and COVID-19 cancer patients. From a functional point of view, genes involved in interferon signaling and cytotoxicity (IFIT1, IFIT3, and MX1) are upregulated in COVID/ cancer cohort. On the other end, genes involved in antigen presentation are downregulated, indicating that in patients with both diseases the inhibition of the immune response is even more pronounced than in patients with only one of the two, suggesting a synergy of action of the two diseases.

GSEA analysis highlighted that the most significantly upregulated pathways between COVID-19 and COVID-19 cancer patients are those induced by interferon. In agreement with the fact that upregulated gene signature in COVID-19 cancer patients is enriched for interferon-regulated genes, their promoters are bound by key regulators of interferon gene expression, IRF, STAT, and BATF TFs. This further supports that. IFN pathways are deregulated in COVID-19 patients [17, 44] . On the other side, IFN pathways are usually active in cancer [45, 46] . Their further induction in COVID-19 cancer patients could indicate a functional synergy between COVID-19 and cancer suggesting that the active IFN pathways in these patients may be one of the factors influencing the increased disease severity observed in COVID 

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We would like to thank the medical directorates of the participating hospitals who made this work possible. We thank the patients who have donated their blood for this study.

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary informations files. NanoString data are available at Gene Expression Omnibus database with access ID GSE164571. AS and LdL acquired, analyzed, and interpreted the data. LdL, CDV, SDM, FDN, FG,  CM, and FP collected and processed the patient samples. MD'A, AR, AT, PM, AN, LDB,  AN, CN, PA, VS, and RM designed and supervised sample collections. DD and BT revised the manuscript. MF, CC, and SS analyzed the data. GB, GP, and GC conceptualized and designed the experiments, acquired the data, and wrote the manuscript. All authors revised and approved the final version of the manuscript.

This work was supported in part by funds Ricerca Corrente from the Ministry of Health, Italy, and by Grant COMETA To GC and RM from Istituto Buddista Italiano Soka Gakkai.

A written informed consent was obtained by all participants to the study. The study was approved by the Central Ethical Committee of IRCCS Lazio -Registro Sperimentazioni n.1446/20 and the Institutional Ethical Committee of University La Sapienza of Rome, Italy (RIF. CE 5773_2020, Prot. # 52SA_2020, and Prot. # 171SA_2020), on the basis that it complied with the declaration of Helsinki and that the protocol followed existing good clinical practice guidelines.

The authors declare no competing interests.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41419-021-04299-y.Correspondence and requests for materials should be addressed to Giovanni Blandino or Giulia Piaggio.

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