Article Text

Single-cell sequencing unveils extensive intratumoral heterogeneity of cancer/testis antigen expression in melanoma and lung cancer
  1. Sofie Traynor1,
  2. Mie K Jakobsen1,
  3. Tina M Green2,
  4. Hana Komic3,4,
  5. Yaseelan Palarasah1,
  6. Christina B Pedersen1,
  7. Henrik J Ditzel1,5,
  8. Fredrik B Thoren3,4,
  9. Per Guldberg1,6 and
  10. Morten F Gjerstorff1,5
  1. 1Department of Cancer and Inflammation Research, Institute for Molecular Medicine, University of Southern Denmark, Odense, Denmark
  2. 2Department of Pathology, Odense University Hospital, Odense, Denmark
  3. 3TIMM Laboratory at Sahlgrenska Center for Cancer Research, University of Gothenburg, Goteborg, Sweden
  4. 4Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Goteborg, Sweden
  5. 5Department of Oncology, Odense University Hospital, Odense, Denmark
  6. 6Danish Cancer Institute, Kobenhavn, Denmark
  1. Correspondence to Dr Morten F Gjerstorff; mgjerstorff{at}health.sdu.dk

Abstract

Cancer/testis antigens (CTAs) are widely expressed in melanoma and lung cancer, emerging as promising targets for vaccination strategies and T-cell-based therapies in these malignancies. Despite recognizing the essential impact of intratumoral heterogeneity on clinical responses to immunotherapy, our understanding of intratumoral heterogeneity in CTA expression has remained limited. We employed single-cell mRNA sequencing to delineate the CTA expression profiles of cancer cells in clinically derived melanoma and lung cancer samples. Our findings reveal a high degree of intratumoral transcriptional heterogeneity in CTA expression. In melanoma, every cell expressed at least one CTA. However, most individual CTAs, including the widely used therapeutic targets NY-ESO-1 and MAGE, were confined to subpopulations of cells and were uncoordinated in their expression, resulting in mosaics of cancer cells with diverse CTA profiles. Coordinated expression was observed, however, mainly among highly structurally and evolutionarily related CTA genes. Importantly, a minor subset of CTAs, including PRAME and several members of the GAGE and MAGE-A families, were homogenously expressed in melanomas, highlighting their potential as therapeutic targets. Extensive heterogeneity in CTA expression was also observed in lung cancer. However, the frequency of CTA-positive cancer cells was notably lower and homogenously expressed CTAs were only identified in one of five tumors in this cancer type. Our findings underscore the need for careful CTA target selection in immunotherapy development and clinical testing and offer a rational framework for identifying the most promising candidates.

  • Skin Cancer
  • Lung Cancer

Data availability statement

Data are available upon reasonable request.

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Introduction

Cancer/testis antigens (CTAs) constitute a unique group of proteins that has attracted considerable attention in cancer research and therapy. In healthy adults, these antigens are restricted to testis germ cells, which are characterized by a state of global DNA hypomethylation, leading to the activation of CTA genes.1 2 As loss of DNA methylation is also a hallmark of cancer development, CTAs are widely expressed across cancer types.1 2

CTAs were originally identified based on their immunogenic properties in tumors. Subsequent investigations have unequivocally demonstrated the ability of a broad selection of these proteins to elicit spontaneous CD4+ and CD8+ T-cell responses and generate cytolytically active T cells capable of targeting cancer cells in patients.3 4 This highlights their inherent immunogenicity and their potential as viable targets for immunotherapeutic interventions.1 2 Consequently, numerous clinical trials have been conducted, with a primary focus on specific CTAs such as NY-ESO-1 and MAGE-A.3 4 These trials have predominantly enrolled patients with melanoma or lung cancer, given the frequent expression of CTAs in these malignancies.5–8

Early strategies focused on vaccination with CTAs, showed promise in inducing cellular immunological responses.9–12 In the largest of these trials, the double-blinded, randomized, MAGRIT phase III study, 2,272 patients with MAGE-A3-positive non-small cell lung cancer (NSCLC) were treated with recombinant MAGE-A3 supplemented with AS15 adjuvant or placebo.9 Unfortunately, this and other CTA vaccination trials fell short in delivering discernible clinical benefits. More recently, adoptive cell transfer (ACT) of T cells engineered to express CTA-specific T-cell receptors has undergone clinical testing.13–20 While some patients exhibited remarkable responses, these effects were often transient or absent in the majority. Thus, despite the promise of CTAs as targets for cancer immunotherapy, there is a need for refined therapeutic approaches capable of overcoming the challenges associated with therapeutic resistance.

Intratumoral heterogeneity represents a critical driver of therapeutic resistance, also in the context of immunotherapeutic strategies. The immune targeting of tumor antigens can inadvertently give rise to the selective outgrowth of antigen-negative immune escape variants within tumors, ultimately resulting in therapeutic relapse.13 17 21 To efficiently address this challenge, it is of paramount importance to carefully select broadly expressed antigen targets to maximize therapeutic efficacy. Previous studies have indicated the heterogeneous expression of CTAs in tumors,6 22–29 posing a substantial challenge to the development of effective vaccines or ACT-based therapeutics targeting specific CTAs within tumors. Despite the potential significance of intratumoral heterogeneity in the efficacy of CTA-based immunotherapies, the intratumoral expression patterns of CTAs have not been comprehensively characterized.

In this study, we employ focused single-cell mRNA sequencing (scRNA-seq) to characterize the intratumoral patterns of CTA expression in melanoma and lung cancer. Our findings reveal an unexpected level of intratumoral complexity in CTA expression, carrying implications for the future design of CTA-based immunotherapies.

Methods

Melanoma and lung cancer tumors

Melanoma cells were isolated from primary and metastatic lesions at the Department of Dermatology, Yale University School of Medicine. Culture conditions were described previously.30 31 The cells were used in passages 3–6. When relevant, cell identities were verified using DNA fingerprinting by short tandem repeat analysis (Cell ID System Promega). Fresh tumor tissue was collected from the primary tumors of five patients with lung adenocarcinoma (LUAD) undergoing standard surgical treatment at Odense University Hospital. A description of tumor dissociation and purification of cancer cells is provided in the online supplemental methods. All patient samples were collected in compliance with the informed consent policy and coded to maintain patient confidentiality. Exempt from informed consent was given by the local ethics committee. Patient data is available in online supplemental table 1.

Supplemental material

Supplemental material

Immunostaining of CTAs in tumors and primary cell cultures

Immunostaining of nine melanoma primary cell cultures and representative melanoma and LUAD tumors was essentially done as previously described.32 Please refer to online supplemental Methods for a list of antibodies.

scRNA-seq analysis of cancer/testis antigen expression in melanoma and lung cancer

The BD Rhapsody system was used to profile the expression of CTAs in seven low-passage melanoma primary cell cultures and isolated cancer cells from five surgical specimens of LUAD tumors. Please refer to online supplemental methods for a detailed description and to online supplemental table 2 for a list of CTAs analyzed by scRNA-seq in melanomas and lung cancers.

Supplemental material

Result

scRNA-seq reveals extensive intratumoral transcriptional heterogeneity of CTA genes in melanoma and lung cancer

Consistent with previous findings,6 22–29 immunohistochemical analysis of selected CTAs (GAGE, MAGE-A, MAGE-C1, NY-ESO-1, PRAME and SSX) revealed a highly variable expression in melanomas (figure 1A and online supplemental figure 1) and lung cancers (online supplemental figure 1). To gain a comprehensive understanding of CTA expression patterns in these malignancies, we conducted targeted CTA scRNA-seq expression profiling of tumor cells. Initially, we examined seven primary cell cultures derived from melanoma tumors, representing a diverse cohort of patients, both male and female, aged 44–85 years (online supplemental table 1). The melanoma cells were collected from primary tumors (YUDOSO, YUPEET, YUTOGS and WW165) and metastatic lesions (YUKSI, YUSIT, YUSIF) harboring different driver mutations (online supplemental table 1) and their identity and purity were verified using a panel of melanocyte markers (online supplemental figure 2). Analysis of a panel of 81 CTA genes revealed a variable expression of CTAs across tumors, with the number of expressed genes ranging from 26 to 37 (figure 1B). Notably, every cancer cell derived from these seven distinct melanomas expressed one or more CTA genes (figure 1C), highlighting the prevalence of CTA expression in melanoma and the potential of these antigens as targets for immune targeting of this disease. However, a noticeable variation in the average number of CTA genes expressed per cell was observed among the tumors, ranging from 7 (YUSIV) to 21 (YUDOSO; figure 1D).

Supplemental material

Figure 1

Intratumoral heterogeneity in the expression of CTAs in melanoma and lung cancer. (A) Immunostaining of selected CTA genes in low passage cancer cell cultures from primary melanomas and metastases (brown). Counterstain: hematoxylin (blue). Representative pictures are shown. Magnification×25. (B–D) scRNA-seq analysis of the expression of 81 CTA genes in low passage cancer cell cultures derived from primary tumors (YUDOSO, YUPEET, YUTOGS, WW165) or metastases (YUKSI, YUSIV, YUSIT) of melanomas. Plots show the number of CTA genes expressed in at least 1% of the cancer cells of each tumor (B) the frequency of CTA-positive cells within each tumor (C) and the frequency of CTA genes expressed in individual cancer cells of each tumor (D). (E–G) scRNA-seq analysis of the expression of 78 CTA genes in cancer cells from primary LUADs of five patients (LUAD1, LUAD2, LUAD3, LUAD4, LUAD5). Plots show the number of CTA genes expressed in at least 1% of the cancer cells of each tumor (E) the frequency of CTA-positive cells within each tumor (F) and the frequency of CTA genes expressed in individual cancer cells of each tumor (G). (H) scRNA-seq analysis of low passage cancer cell cultures derived from melanoma primary tumors and metastases. The dot plot shows the frequency and intensity (average expression) of CTA genes expressed in >10% of cells in at least one tumor. (I) scRNA-seq analysis of cancer cells from primary LUAD tumors of five patients. The dot plot shows the frequency and intensity (average expression) of CTA genes expressed in >10% of cells in at least one tumor. (J) Expression frequency of selected CTA genes among cancer cells of melanomas and LUADs. The expression frequencies of all expressed CTA genes are shown in online supplemental figure S1 and S3. CTA, cancer/testis antigens; LUAD, lung adenocarcinoma; scRNA-seq, single-cell messenger RNA sequencing.

There was no discernible difference in the average number of CTAs expressed between primary tumors and metastases (figure 1D). Within each tumor, the average number of CTA genes expressed in individual cells was significantly lower than the total number of CTA genes expressed in the tumor (figure 1B,D). Consistent with this, the expression of individual CTA genes was generally confined to subpopulations of cancer cells (figure 1H and online supplemental figure 3). For example, the prominent tumor target NY-ESO-1 (encoded by CTAG1B) was detected in three tumors (YUDOSO, YUTOGS and YUSIT) out of seven, with frequencies of positive cells at 1%, 48%, and 66% (figure 1J). Similarly, other CTAs with recognized therapeutic potential, such as SSX1, MAGE-A1, MAGE-A10, MAGE-C2, XAGE-1B and PAGE-1, also displayed substantial intratumoral heterogeneity in their expression (figure 1H,J and online supplemental figure 3). Overall, 64 out of 71 detected CTA genes consistently exhibited expression confined to subpopulations of tumor cells.

The remaining seven CTAs showed varying degrees of homogeneity in their expression across melanoma tumors, as illustrated in figure 1H,J and online supplemental figure 1. Particularly noteworthy was PRAME, which exhibited uniform expression in six of the seven melanoma primary tumor cultures (figure 1H,J). Furthermore, MAGEA2, MAGEA3, MAGEA6, and MAGEA12, constituting an evolutionarily conserved subcluster of MAGE-A genes, exhibited homogeneous expression in a subset of metastatic tumors (YUSIT and YUKSI; figure 1H,J and online supplemental figure 3). GAGE2 and GAGE12 were also homogeneously expressed in a subset of melanomas (YUDOSO and YUKSI) and were generally widely expressed in the examined melanomas (figure 1H,J and online supplemental figure 3). The intratumoral patterns of CTA gene expression, as uncovered through scRNA-seq analysis, were largely consistent at the protein level, as demonstrated through immunostainings of selected CTAs, including GAGE, MAGE-A, MAGE-C1, NY-ESO-1 and PRAME (figure 1A). This consistency strongly suggests that the observed differences in CTA expression between cancer cells is not due to temporal variations in gene transcription.

Subsequently, we characterized the intratumoral CTA expression patterns in primary LUAD from five patients, aged 54–8 years, representing both sexes (online supplemental table 1). Notably, the five LUAD tumors exhibited different molecular aberrations (online supplemental table 1). Cancer cells isolated from these tumors underwent focused scRNA-seq to analyze CTA gene expression. To confirm the purity of cancer cells, marker genes for different types of normal lung cells were included in the analysis (online supplemental figure 4). Among the 78 CTA genes analyzed, between 4 and 36 were expressed in the five tumors, respectively (figure 1E). In contrast to the melanoma tumors, there was little overlap in the set of genes activated in the different tumors (figure 1I). The prevalence of CTA expression in LUAD was comparatively more limited than that in melanoma, as the proportion of cells expressing at least one CTA was 100%, 98%, 65% 50% and 21%, respectively (figure 1F). Each cancer cell expressed only a minor subset of the CTA genes expressed by the whole LUAD tumor (figure 1E,G), and most CTAs were limited to subpopulations of cancer cells in tumors (figure 1I,J; online supplemental figure S5). Uniform expression of CTA genes, including GAGE, PRAME and GTSF1, was only observed in a single LUAD tumor (LUAD5; figure 1I and online supplemental figure 5). XAGE-1B, a proposed target in LUAD,33–36 was expressed in up to 84% of the cells, whereas BAGE-2 and PAGE-5 were expressed in up to 41% and 26% of the cells, respectively (online supplemental figure 3). The remaining CTAs, including NY-ESO-1 and MAGE-A family members, were found to be expressed in less than 20% of the cancer cells within the tumors (figure 1I,J and online supplemental figure 5).

Overall, these findings unveiled a remarkably high level of transcriptional heterogeneity among CTA genes in melanoma and LUAD tumors, with uniform CTA expression being a rare phenomenon observed only found in melanoma.

Lack of coordination of CTA expression in tumors generates mosaics of tumor cells with diverse cancer/testis antigen profiles

The co-expression of CTAs has been demonstrated in tumors across cancer types.8 37 To further characterize the landscape of CTA gene expression within melanoma and LUAD tumors and identify potential associations in CTA expression, we conducted a UMAP dimensional reduction analysis of the scRNA-seq data. This analysis identified cellular subsets with differential expression of CTAs and some correlation between the expression of specific CTAs (Figure 2A-E). Nevertheless, these subsets displayed poor separation and a significant overlap in CTA expression, indicating a lack of robust transcriptional disparities and coordinated CTA expression among cancer cells within the same tumor. Indeed, minimal overlap in the expression of distinct CTA gene families was observed in both melanoma and LUAD tumors, consistent with expectations based on chance (figure 3A). Even among distantly-related CTAs, such as those encoding the different types of MAGE families, or CTAs expressed during the same stages of male germ cell maturation (eg, GAGE, MAGE-A, CTAG1B and SSX in spermatogonia),6 23 27 38 co-expression was lacking. This suggested a high degree of variation in the CTA expression profiles among cancer cells within tumors, indicating that melanomas and lung cancers are composed of complex mosaics of tumor cells with diverse antigen profiles. Consistent with this, a direct comparison of the CTA expression profiles of individual cancer cells demonstrated a very high level of diversity (figure 3B,C). Lung cancers were less complex in their CTA expression than melanomas reflecting the lower prevalence of CTA expression (figure 3B,C).

Figure 2

Lack of strong transcriptional disparities in CTA gene expression among cancer cell subsets within tumors. (A–D) Clustering analysis of single-cell CTA gene expression in representative melanomas: YUDOSO (A) YUPEET (B) and YUSIV (C) and LUAD: LUAD1 (D) and LUAD5 (E). Left: UMAP dimensional reduction plots showing identified cell clusters within tumors. Middle: dot plots showing the frequency and relative expression levels of CTA genes with expression >10% in at least one cluster. The fraction of cells within each cluster is indicated. Right: feature plots showing the expression (log1p) of selected CTA genes in identified cell clusters. CTA, cancer/testis antigens; LUAD, lung adenocarcinoma; UMAP, Uniform Manifold Approximation and Projection.

Figure 3

Lack of co-expression among distinct CTA gene families in tumors generates mosaics of cancer cells with diverse antigen profiles. (A–C) Single-cell CTA gene expression in representative melanoma and LUAD tumors. Data for selected tumors are shown. (A) Venn diagrams depict the intratumoral overlap in the expression of selected CTA genes of different gene families. (B) Heat maps show CTA gene expression profiles of individual cancer cells. Gene not expressed=0. Gene expressed=1. Clustering analysis was performed to unveil cancer-cell subsets exhibiting comparable gene expression profiles. (C) Principal component analysis of single cells based on the expression of CTA genes. CTA, cancer/testis antigens; LUAD, lung adenocarcinoma.

To comprehensively investigate potential transcriptional associations among CTAs, we conducted pairwise comparisons of their expression patterns (figure 4A). While the majority of CTAs displayed no significant correlation in their expression, distinct subsets exhibited co-expression within tumors. This intriguing correlation was particularly evident among highly evolutionarily and structurally linked genes, suggesting potential regulation by shared transcription factors or preservation within the same regulatory compartments. For example, MAGEA2, MAGEA3, MAGEA6, and MAGEA12, forming a distinct gene cluster of MAGE-A genes on the X chromosome (figure 4B), demonstrated a remarkable level of overlapping expression among YUPEET tumor cells (figure 4C,D), which was not shared by other MAGE genes (figure 4E). A similar pattern was observed for MAGEA2, MAGEA3, MAGEA6, and MAGEA12 in other tumors (figure 4F) and with other-related CTA genes, such as members of the PAGE family (figure 4G). Infrequent co-expression was also noted among genes from different gene families (figure 4A). Importantly, the identified correlations among CTA-gene subsets were not consistent across tumors. This divergence can be attributed to disparities in chromatin organization among tumors, leading to variations in the regulatory compartments governing CTA genes.

Figure 4

Co-activation of structurally and evolutionary linked CTA genes. (A) Pairwise co-expression analysis of CTA genes in selected melanomas and LUADs. Scales depict the level of co-expression (observed co-expression divided by expected co-expression based on the frequency of expression within tumors). (B) Schematic representation of MAGEA gene family genes on chromosome X. The MAGEA2, MAGEA3, MAGEA6 and MAGEA12 subcluster is highlighted in green. (C) Feature plots of genes belonging to the MAGEA2, MAGEA3, MAGEA6 and MAGEA12 subcluster. Yellow indicates cells with co-expression of indicated genes. (D) Venn diagram depicting overlap in expression of the MAGEA2, MAGEA3, MAGEA6 and MAGEA12 subcluster in the Yupeet melanoma tumor. (E) Venn diagram depicting overlap in expression of distantly-related MAGE genes. (F) Venn diagram depicting overlap in expression of the MAGEA2, MAGEA3, MAGEA6 and MAGEA12 subcluster in the LUAD1 tumor. (G) Venn diagram depicting overlap in expression of PAGE genes in the LUAD2 tumor. CTA, cancer/testis antigens; LUAD, lung adenocarcinoma.

Our findings underscore a remarkable level of complexity in the expression of CTAs in melanomas and lung cancers. The general absence of co-expression of CTAs with coordinated expression in spermatogenesis suggests a lack of bona fide germ cell transcriptional programs in tumors, contrary to previous proposals.1 Instead, it suggests that the activation of CTA genes in cancer cells appears to be largely guided by stochastic processes.

Discussion

Our study unveils the highly variable nature of CTA expression profiles within tumors. Most CTAs exhibited expression limited to distinct and minimally overlapping subsets of cancer cells, generating mosaics of cancer cells with diverse CTA profiles. The heterogeneity in CTA expression has implications for their value as therapeutic targets. An illustrative example is NY-ESO-1 (encoded by CTAG1B), an appealing therapeutic target due to its frequent expression in tumors and its ability to elicit robust humoral and cellular responses.4 Several clinical trials have employed vaccines or genetically modified T cells targeting NY-ESO-1 to treat metastatic cancers, including melanoma and NSCLC, with promising outcomes.4 Despite these efforts, the development of resistance and clinical progression remains a major obstacle, partly attributed to the selective loss of NY-ESO-1 expression within tumors.13 17 Our comprehensive analysis of seven melanomas and five LUADs using scRNA-seq antigen profiling revealed substantial intratumoral heterogeneity in NY-ESO-1 expression, which may explain the limited long-term benefits observed in clinical trials targeting this antigen. It further suggests that NY-ESO-1 is unsuitable as a stand-alone therapeutic target.

In addition to NY-ESO-1, MAGE-A3 represents a frequently pursued CTA target in both past and ongoing clinical trials,39 with the most comprehensive being the MAGRIT Phase III trial targeting MAGE-A3 in NSCLC. Our results demonstrated MAGE-A3 expression in two of five LUAD tumors, a subset of NSCLC, but in only 0.5% and 18% of the cancer cells, respectively. This observed intratumoral heterogeneity in MAGE-A3 expression raises the possibility that it may have contributed to the discouraging outcomes of the MAGRIT trial.9 Moreover, it could pose a formidable challenge for ongoing and future clinical trials targeting this specific CTA. Intratumoral heterogeneity in expression was also observed for other CTA targets currently undergoing clinical development, raising questions about their therapeutic value.

Notably, some CTAs displayed homogeneous expression in subsets of melanoma tumors. In particular, PRAME exhibited highly uniform expression in six of seven melanomas, as demonstrated by both scRNA-seq and immunostaining, suggesting that targeting PRAME may have the potential to achieve complete responses to treatment. Currently, PRAME is the target of several clinical trials for the treatment of metastatic cancer. GAGE and MAGE-A family members also showed uniform expression in subsets of melanomas and broad expression in others. These findings indicate that PRAME, GAGE, and MAGE-A antigens represent the most promising CTA candidates for immunotherapy in the context of melanoma, based on their prevalence within tumors.

The diversity in CTA profiles among cancer cells within tumors, as demonstrated in this study, strongly supports the advancement of immunotherapeutic strategies with broad targeting approaches. The lack of co-expression among different CTA gene families should be considered in designing strategies to maximize the percentage of targetable cancer cells in tumors. Multitarget vaccines, customized based on data from scRNA-seq CTA profiling, represent a potential avenue for achieving this goal. In melanoma, broadly reactive CTA-directed therapies hold the potential to target all cancer cells, given their expression of at least one CTA. In contrast, our analysis of LUAD tumors revealed CTA-negative subpopulations in four of five tumors, suggesting that even therapies targeting multiple CTAs may not provide complete responses in the majority of patients with this disease.

In conclusion, while CTAs remain promising targets for cancer immunotherapy, their heterogeneous expression within tumors may contribute to the lack of sustained therapeutic benefits observed in many patients. Accurately quantifying the variation in intratumoral expression frequencies of specific CTA genes among melanomas and LUADs requires a larger panel of tumors. Our data show that further research and careful consideration of CTA tumor heterogeneity, as is done for therapeutic neoantigens, are necessary to devise more effective and lasting immunotherapeutic strategies.

Data availability statement

Data are available upon reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

This study involves human participants and was approved by Ethics Committee of the Region of Southern Denmark (S20230029). Exempt from informed consent was given by the local ethics committee.

Acknowledgments

We thank Lone Christiansen at the Department of Pathology, Odense University Hospital for technical assistance with immunohistochemistry.

References

Supplementary materials

Footnotes

  • X @Thoren_lab

  • ST and MKJ contributed equally.

  • Contributors Conceptualization: ST, MJ and MFG. Methodology: ST, MJ, TMG, HK, YP, CBP, HJD, FBT, PG and MFG. Investigation: ST, MJ, TMG, HK, YP, CBP, FBT and MFG. Writing—original draft: MFG. Critical revision of the manuscript: ST, MJ, TMG, HK, HJD, FBT, PG and MFG. Writing—review and editing: all authors. Funding acquisition: MJ, ST and MFG. Supervision: MFG.

  • Funding The study was supported by the Region of Southern Denmark, Pink Tribute, Einar Willumsens foundation, Carl and Ellen Hertzs foundation, Phycician Sofus Carl Emil Friis and spouse’s foundation, Neye foundation, Agnes and Poul Friis foundation, Tornoes og Hoyrups foundation, Emil C. Hertz and spouse’s foundation, Frimodt-Heineke foundation, Olga Doris Friis foundation, Dagmar Marshalls foundation and Danish Cancer Society (R1R204-A12255).

  • Competing interests No, there are no competing interests.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.