Cutaneous T-Cell Lymphoma Skin Microbiome Is Characterized by Shifts in Certain Commensal Bacteria but not Viruses when Compared with Healthy Controls

Abstract

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphoid malignancies derived from skin-homing T cells. Mycosis fungoides (MF) is the most common form of CTCL, and Sezary syndrome (SS) is an aggressive variant with varying levels of clonal lymphocytes in the blood. The variable presentation and lack of definitive diagnostic markers make CTCL diagnosis challenging. Although the biology of these malignancies is not fully understood, some microbes, particularly viruses, have been hypothesized to play roles in malignant T-cell transformation in CTCL (Berger et al., 2002Berger C.L. Hanlon D. Kanada D. Dhodapkar M. Lombillo V. Wang N. et al.The growth of cutaneous T-cell lymphoma is stimulated by immature dendritic cells.Blood. 2002; 99: 2929-2939Crossref PubMed Scopus (110) Google Scholar; Mirvish et al., 2013Mirvish J.J. Pomerantz R.G. Falo L.D. Geskin L.J. Role of infectious agents in cutaneous T-cell lymphoma: facts and controversies.Clin Dermatol. 2013; 31: 423-431Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar; van der Loo et al., 1979van der Loo E.M. van Muijen G.N. van Vloten W.A. Beens W. Scheffer E. Meijer C.J. C-type virus-like particles specifically localized in Langerhans cells and related cells of skin and lymph nodes of patients with mycosis fungoides and Sezary's syndrome. A morphological and biochemical study.Virchows Arch B Cell Pathol Incl Mol Pathol. 1979; 31: 193-203Crossref PubMed Scopus (69) Google Scholar). However, high throughput sequencing approaches have failed to consistently detect viral sequences in the skin or peripheral blood of patients with CTCL (Anderson et al., 2018Anderson M.E. Nagy-Szakal D. Jain K. Patrone C.C. Frattini M.G. Lipkin W.I. et al.Highly sensitive virome capture sequencing technique VirCapSeq-VERT identifies partial noncoding sequences but no active viral infection in cutaneous T-cell lymphoma.J Invest Dermatol. 2018; 138: 1671-1673Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar; Dereure et al., 2013Dereure O. Cheval J. Du Thanh A. Pariente K. Sauvage V. Claude Manuguerra J. et al.No evidence for viral sequences in mycosis fungoides and Sezary syndrome skin lesions: a high-throughput sequencing approach.J Invest Dermatol. 2013; 133: 853-855Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Infections are common in patients with advanced stage, and antibiotic treatment results in skin improvement and decreased disease activity (Lindahl et al., 2019Lindahl L.M. Willerslev-Olsen A. Gjerdrum L.M.R. Nielsen P.R. Blümel E. Rittig A.H. et al.Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.Blood. 2019; 134: 1072-1083Crossref PubMed Scopus (64) Google Scholar). Hence, to better understand the spectrum of microbial involvement in CTCL, we performed a comprehensive evaluation of the skin microbiome in a cohort of patients with MF and SS as compared with healthy controls. In this pilot study, we used shotgun metagenomic sequencing to investigate microbial communities at predetermined, matched skin sites in 4 patients with MF (stages IA–IIIA), 2 patients with SS (stage IVA1), and 10 age- and sex-matched healthy volunteers (HVs) (Supplementary Table S1). The study was approved by the Institutional Review Boards of Johns Hopkins (Baltimore, MD) and the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH (Bethesda, MD). Subjects provided written informed consent and underwent skin preparatory regimens. Premoistened swabs were used to collect samples from the nares, lower back, and thigh skin (sites of CTCL predilection) and air controls (see Supplementary Materials and Methods for details on patient recruitment and sampling). DNA was isolated, and libraries were created for metagenomic sequencing (Oh et al., 2014Oh J. Byrd A.L. Deming C. Conlan S. NISC Comparative Sequencing Program Kong H.H. et al.Biogeography and individuality shape function in the human skin metagenome.Nature. 2014; 514: 59-64Crossref PubMed Scopus (572) Google Scholar). Bacterial, fungal, and viral communities were investigated by mapping microbial reads to a multikingdom reference database (Supplementary Table S2). Analyses focused on comparing the microbiomes between lesional patient skin and HV skin from the lower back and thigh. Bacteria predominated microbial communities at all sites (Figure 1a, Supplementary Figures S1a and S2a, and Supplementary Table S3). Of the most abundant taxa across kingdoms (Figure 1b and Supplementary Figures S1b and S2b), <0.5% of metagenomic reads mapped to eukaryotic viruses (predominantly Papillomaviridae and Polyomaviridae) in MF and/or SS and HV lower backs (0.09% ± 0.1% vs. 0.05% ± 0.05%) and thighs (0.08% ± 0.1% vs. 0.07% ± 0.08%) with no discernible differences regardless of sampling area (Figure 1c, Supplementary Figures S1c and S2c, and Supplementary Table S4). Similarly, fungal abundances did not differ significantly between HVs and patients with MF and/or SS (Figure 1b, Supplementary Figure S1b, and Supplementary Table S5); Shannon diversity was comparable (Supplementary Figure S3a and b). Given the low viral and fungal relative abundances, we focused on bacterial communities in patients with MF and/or SS and HVs. We performed principle coordinates analysis using Bray-Curtis dissimilarity index, which demonstrated separation of HV and MF and/or SS bacterial skin communities on both lower backs and thighs (Figure 1d and Supplementary Figure S1d). Superimposing MF and/or SS stages on the principle coordinates analysis showed greatest separation between HVs and patients with stage IVA1, suggesting that skin microbiomes of patients with stage IV are the most distinct from controls. We then further investigated specific taxa contributing to differences in bacterial communities among MF, SS, and HV skin. Given the association with Staphylococcus aureus colonization and infection in CTCL (Krejsgaard et al., 2014Krejsgaard T. Willerslev-Olsen A. Lindahl L.M. Bonefeld C.M. Koralov S.B. Geisler C. et al.Staphylococcal enterotoxins stimulate lymphoma-associated immune dysregulation.Blood. 2014; 124: 761-770Crossref PubMed Scopus (42) Google Scholar; Lindahl et al., 2019Lindahl L.M. Willerslev-Olsen A. Gjerdrum L.M.R. Nielsen P.R. Blümel E. Rittig A.H. et al.Antibiotics inhibit tumor and disease activity in cutaneous T-cell lymphoma.Blood. 2019; 134: 1072-1083Crossref PubMed Scopus (64) Google Scholar) and reported staphylococcal-corynebacterial interactions (Ramsey et al., 2016Ramsey M.M. Freire M.O. Gabrilska R.A. Rumbaugh K.P. Lemon K.P. Staphylococcus aureus shifts toward commensalism in response to Corynebacterium species.Front Microbiol. 2016; 7: 1230Crossref PubMed Scopus (133) Google Scholar), we compared these and other common cutaneous bacteria. S. aureus abundances were low in most MF, SS, and HV skin samples (Supplementary Figure S4a and b). One HV and one patient with MF had higher S. aureus relative abundances on the skin. Commensal staphylococci (S. capitis, S. epidermidis, and S. hominis) trended higher in MF (3.8% ± 3.9%, 2.7% ± 2.1%, and 1.8% ± 2.4%, respectively) versus HV lower back skin (0.6% ± 0.6%, 1.4% ± 1.1%, and 0.8% ± 1.2%, respectively) (Supplementary Figure S4). Two Corynebacterium species (C. tuberculostearicum and C. simulans) were increased on MF and SS skin, with highest mean relative abundances in patients with SS (C. tuberculostearicum on lower back: 25.6% ± 24.3% [SS] vs. 4.4% ± 5.8% [HV]; C. simulans on lower back: 6.5% ± 5.5% [SS] vs. 0.3% ± 0.5% [HV]) (Figure 1e and Supplementary Figure S1e). MF and SS skin also displayed lower relative abundances of Cutibacterium acnes and Cutibacterium namnetense than HV skin. These bacterial shifts were not statistically significant, likely because of the small number of patients. However, comparing HV to MF to SS skin, we observed increasing trends in the mean relative abundances of Corynebacterium species and decreasing trends in Cutibacterium species, suggesting that bacterial shifts may correlate with disease stage or treatment status (Figure 1e and Supplementary Figure S1e). Our findings suggest that eukaryotic DNA viruses are negligible components of the skin microbiome in our MF and/or SS cohort. These results corroborate and extend previous reports suggesting that CTCL is unlikely to originate from infection by a directly oncogenic DNA virus (Dulmage et al., 2015Dulmage B.O. Feng H. Mirvish E. Geskin L. Black cat in a dark room: the absence of a directly oncogenic virus does not eliminate the role of an infectious agent in cutaneous T-cell lymphoma pathogenesis.Br J Dermatol. 2015; 172: 1449-1451Crossref PubMed Scopus (10) Google Scholar). However, other mechanisms by which viral pathogens can affect neoplastic transformation must be further explored including (i) indirect viral tumorigenesis by transient exposure to viral genomes (hit-and-run oncogenesis) (Niller et al., 2011Niller H.H. Wolf H. Minarovits J. Viral hit and run-oncogenesis: genetic and epigenetic scenarios.Cancer Lett. 2011; 305: 200-217Crossref PubMed Scopus (79) Google Scholar), (ii) integration of retroviruses or DNA viral elements into human host DNA, and (iii) antigenic stimulation of T cells in the peripheral blood. Our patients with MF and/or SS showed no marked differences in skin viral or fungal communities as compared with age-matched HVs sampled at consistent sites. Nonetheless, we observed bacterial community shifts including higher relative abundances of Corynebacterium species and lower relative abundances of Cutibacterium species. in MF and/or SS skin. Several staphylococcal and corynebacterial species trended higher in MF and/or SS skin and would be important to examine in larger studies. Relative abundances of C. tuberculostearicum were high (>25% on average) in patients with stage IVA1. Patients with advanced stage may be at increased risk of infection from impaired skin integrity and immune dysregulation (Axelrod et al., 1992Axelrod P.I. Lorber B. Vonderheid E.C. Infections complicating mycosis fungoides and Sézary syndrome.JAMA. 1992; 267: 1354-1358Crossref PubMed Scopus (178) Google Scholar), and C. tuberculostearicum has been shown to upregulate proinflammatory responses in human skin cells, suggesting a potential link to cutaneous inflammation (Altonsy et al., 2020Altonsy M.O. Kurwa H.A. Lauzon G.J. Amrein M. Gerber A.N. Almishri W. et al.Corynebacterium tuberculostearicum, a human skin colonizer, induces the canonical nuclear factor-kB inflammatory signaling pathway in human skin cells.Immun Inflamm Dis. 2020; 8: 62-79Crossref PubMed Scopus (7) Google Scholar). Recently, Salava et al., 2020Salava A. Deptula P. Lyyski A. Laine P. Paulin L. Väkevä L. et al.Skin microbiome in cutaneous T-cell lymphoma by 16S and whole genome shotgun sequencing.J Invest Dermatol. 2020; 140: 2304-2308.e7Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar reported some differences in bacterial communities between clinically unaffected and affected patient skin without including HV controls. Because of the potential absence of uninvolved contralateral skin in patients with advanced stage, reports of lymphoma involvement in normal-appearing skin in patients with CTCL (Pujol et al., 2000Pujol R.M. Gallardo F. Llistosella E. Blanco A. BernadóL Bordes R. et al.Invisible mycosis fungoides: a diagnostic challenge.J Am Acad Dermatol. 2000; 42: 324-328Abstract Full Text Full Text PDF PubMed Google Scholar), and different skin microbiomes in anatomically distinct but adjacent sites, we studied control samples from age-matched HVs sampled at comparable anatomical sites. The separation of bacterial communities between patients with stage IVA1 and HVs is notable, and it is intriguing that bacterial shifts appear to correlate with disease stage. Whether these findings are driven by disease severity or other unrelated variables, including systemic treatments, is unknown. Further investigation with larger and ideally multicenter CTCL cohorts is warranted to validate these findings and to evaluate the relationship between disease stage and the skin microbiome. All sequencing data were deposited and are available at the National Center for Biotechnology Information Sequence Read Archive under BioProject number PRJNA642893. Catriona P. Harkins: http://orcid.org/0000-0002-9099-7291 Margaret A. MacGibeny: http://orcid.org/0000-0002-3353-7176 Katherine Thompson: http://orcid.org/0000-0003-3532-0779 Bianka Bubic: http://orcid.org/0000-0001-7916-1349 Xin Huang: http://orcid.org/0000-0003-2466-4373 Isabelle Brown: http://orcid.org/0000-0002-9313-7895 Jin Park: http://orcid.org/0000-0002-8830-5479 Jay-Hyun Jo: http://orcid.org/0000-0002-2039-3425 Julia A. Segre: http://orcid.org/0000-0001-6860-348X Heidi H. Kong: http://orcid.org/0000-0003-4424-064X Sima Rozati: http://orcid.org/0000-0001-6318-6429 The authors state no conflict of interest. The authors thank other members of the Segre and Kong labs for their underlying efforts and the healthy volunteers and patients with cutaneous T-cell lymphoma for their contributions. This publication was supported by the Intramural Research Programs of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (CPH, MAM, JP, JHJ, HHK) and the National Human Genome Research Institute (CPH, XH, JAS). The study utilized the computational resources of the National Institutes of Health High Performing Computing Biowulf Cluster (http://hpc.nih.gov). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Conceptualization: JAS, HHK, SR; Data Curation: CPH, MAM, KT, BB, XH, IB, SR; Formal Analysis: CPH, XH; Funding Acquisition: JAS, HHK, SR; Investigation: CPH, KT, BB, XH, IB, JP, JHJ, SR; Methodology: JAS, HHK, SR; Project Administration: HHK, SR; Resources: JAS, HHK, SR; Software: CPH, XH; Supervision: JAS, HHK, SR; Validation: CPH, XH, JAS, HHK, SR; Visualization: CPH, MAM, XH; Writing - Original Draft Preparation: MAM, HHK, SR; Writing - Review and Editing: CPH, MAM, XH, JP, JHJ, JAS, HHK, SR Download .pdf (.36 MB) Help with pdf files Supplementary Materials Download .xlsx (1.76 MB) Help with xlsx files Supplementary Tables