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Clonality, Mutation and Kaposi Sarcoma: A Systematic Review

Simple Summary Kaposi’s sarcoma (KS) is a rare tumour of uncertain nature. It may be a true cancer or an aggressive viral lesion, which can regress in many patients, and there is a real need for more research on the subject. This systematic review aimed to summarize the available evidence on somatic mutations and clonality within KS to assess whether KS is a neoplasm or not, concluding that knowledge is currently insufficient to determine whether KS is a clonal neoplasm (sarcoma), or simply an aggressive reactive virus-driven lesion. Abstract Background: It remains uncertain whether Kaposi sarcoma (KS) is a true neoplasm, in that it regresses after removal of the stimulus to growth (as HHV8) when immunosuppression is reduced. We aimed to summarize the available evidence on somatic mutations and clonality within KS to assess whether KS is a neoplasm or not. Methods: Medline and Web of Science were searched until September 2020 for articles on clonality or mutation in KS. Search strings were supervised by expert librarians, and two researchers independently performed study selection and data extraction. An adapted version of the QUADAS2 tool was used for methodological quality appraisal. Results: Of 3077 identified records, 20 publications reported on relevant outcomes and were eligible for qualitative synthesis. Five studies reported on clonality, 10 studies reported on various mutations, and 5 studies reported on chromosomal aberrations in KS. All studies were descriptive and were judged to have a high risk of bias. There was considerable heterogeneity of results with respect to clonality, mutation and cytogenetic abnormalities as well as in terms of types of lesions and patient characteristics. Conclusions: While KS certainly produces tumours, the knowledge is currently insufficient to determine whether KS is a clonal neoplasm (sarcoma), or simply an aggressive reactive virus-driven lesion.


Introduction
Kaposi sarcoma (KS) is an unusual neoplasm, which presents with clinical manifestations that range from those that are slowly progressive and confined to the skin, to an aggressive tumour that invades visceral organs. The clinicopathological characteristics are usually described in terms of the location of the lesions (lymph node, visceral or skin), and the clinical stage of the disease (patch, plaque or nodular). Epidemiologically, KS presents in several forms: (a) a classic form predominantly in older men; (b) an endemic form in young men and children from central Africa; (c) an iatrogenic form secondary to treatment with immunosuppressive drugs, such as steroids; and (d) an epidemic, HIV/AIDS-associated form [1].
in his studies of retinoblastoma [16]. However recent research is broadening our knowledge, and many studies suggest that cancer develops as a result of somatic mutation and clonal selection [11,17,18]. The high frequency of cancer-driving mutations in normal tissues sometimes appears to indicate that somatic mutation and clonal selection alone are insufficient to explain cancer development and that other factors must be required to promote carcinogenesis.
KS is currently classified as a neoplasm by the WHO Classification of Tumours (WCT); however, this constitutes a controversial topic that continues to be reviewed periodically. Any future decision on its status will be informed by the best available evidence. The aim of this systematic review is to summarize any available published scientific evidence on KS with regard to somatic mutations and clonality in order to determine whether KS can be defined as a neoplasm or not. The results of this evidence synthesis will serve to inform future decisions of the WCT and detect possible evidence gaps.

Materials and Methods
A systematic review was conducted to identify peer-reviewed articles of data on clonality or somatic mutations in KS following a protocol registered with PROSPERO (CRD42018087595). This review was performed following the PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines and considering the recently published update of these guidelines in 2020 [19].

Literature Search and Study Selection
Tailored conceptual strings of relevant keywords and database-specific terms were devised for the major concepts of Kaposi Sarcoma, Clonality and mutations. Conceptual strings were combined with Boolean operators using appropriate MeSH headings and filters to search the Medline and Web of Science databases for peer-reviewed articles published up to 01 September 2020, without date restrictions. The Cochrane library was also consulted, and the reference lists of relevant articles were checked for additional studies. The concepts and keywords used to inform the search strategy are detailed in Table 1, and the full search strategy is presented in Supplementary Materials S1. Studies were excluded if they exclusively focused on: (1) Kaposi Sarcoma Herpes Virus (KSHV) mutations or viral clonality; (2) on cell lines/cultures; (3) describing tumour phenotype, proteins, receptors, vascular markers etc.; (4) mitochondrial genomes; or (5) the epidemiology or aetiology of KS; or if they were not original research, such as editorials, letters, narrative reviews and book chapters. Titles and abstracts were screened by two reviewers independently (I.C. and B.I.I.), and the full text of potentially eligible articles was assessed to decide final inclusion by both of them. The lists of included articles were compared and differences about the selected studies were resolved by consensus.

Data Extraction
All data were extracted by two researchers independently (B.I.I. and A.L.) into standardized data extraction forms. Discrepancies in data extraction were resolved by discussion and consensus. Relevant information was recorded from the selected publications, including the author(s), publication year, country where the study was performed, baseline population characteristics and demographics, study methodology, methods for clonality or mutation testing, main results on clonality, somatic mutations or chromosomal aberrations (only descriptive outcomes due to the nature of the studies) as well as COI disclosure. We registered frequency measures and ordered the rest of outcomes strategically in order to facilitate interpretation of the results. Data were recorded and compiled using Microsoft Excel.

Assessment of Risk of Bias of Included Studies
Risk of bias of included individual studies was performed using an ad hoc adapted version of the QUADAS2 tool for the Quality Assessment of Diagnostic Accuracy Studies [20]. This assessment included a general appraisal of the external and internal validity of the selected studies, as well as of the biases relevant to studies focusing on diagnostic determinations, adapted ad hoc to the retrieved study designs. One reviewer (B.I.I.) scored items as low, high, or unclear risk of bias; the results were discussed with the review team and resolved by consensus. The final evaluation of all studies was included into the summary of findings tables after resolving differences through arbitration of a third reviewer (I.A.C.).

Synthesis of Results
The results were tabulated in Excel sheets and summary of findings tables were drafted to present the main outcomes for each of the included studies. Outcomes were grouped based on their focus: clonality, somatic mutations or chromosomal aberrations. A descriptive analysis was performed for a qualitative synthesis of the results. The performance of a meta-analysis to calculate an adjusted pooled estimate was ruled out because the descriptive nature of the retrieved studies means that a pooled summary of the data would not be useful. Additional important methodological heterogeneity between the studies from the reported outcome measures rendered formal quantitative synthesis inappropriate. Instead, the aim was to narratively assess and combine the studies in order to derive clinically meaningful conclusions about the nature of KS.

Results
Of the 3077 records identified and screened through the database searches, 25 publications were eventually deemed as relevant and retrieved for full text inspection and qualitative synthesis ( Figure 1). Five of these records were conference abstracts and were therefore excluded for not being peer-reviewed research.
Results of the included studies are summarised in Tables 2-4 according to the following outcome categories: (case series) reporting outcomes on clonality, reporting on mutations and reporting on chromosomal aberrations. After applying the eligibility criteria, 5 studies reporting on clonality, 10 studies on various mutations and 5 studies reporting on chromosomal aberrations in KS were selected. The majority of publications were traditional case series; however, two of the selected studies aimed applied a different study design.
Tornesello et al. [21] performed a case control study investigating TP53 mutations in classic, epidemic and endemic KS cases, and Cerimele et al. [22] applied a prospective design to a case series obtained from a large cohort study in Sardinia. Nevertheless, only descriptive analysis of the genetic factors was provided by these studies as with the other case series, since the main aim of the studies relied on investigating the epidemiology of endemic cases. Studies provided descriptive results with frequency measures, and only a few studies provided analysis of the differences between groups. Results of the included studies are summarised in Tables 2-4 according to the following outcome categories: (case series) reporting outcomes on clonality, reporting on mutations and reporting on chromosomal aberrations. After applying the eligibility criteria, 5 studies reporting on clonality, 10 studies on various mutations and 5 studies reporting on chromosomal aberrations in KS were selected. The majority of publications were traditional case series; however, two of the selected studies aimed applied a different study design.
Tornesello et al. [21] performed a case control study investigating TP53 mutations in classic, epidemic and endemic KS cases, and Cerimele et al. [22] applied a prospective design to a case series obtained from a large cohort study in Sardinia. Nevertheless, only The studies were performed using data from 13 different countries, and eight were international collaborations (Tables 2-4). The first study was published in 1984, and the most recent one was from 2015. Together, the studies enrolled 498 cases of KS and 421 controls; 46 cases and 12 controls to investigate clonality, 238 cases and 408 controls to study mutations and 214 cases and 1 control for chromosomal aberration studies. Studies on clonality included only female cases and were of poor reporting quality; often not reporting basic demographic variables, such as age. Additionally, the studies on mutations and chromosomal aberrations often did not provide adequate patient population and sample descriptions, making it difficult to assess the representativeness of the samples (Summary of Findings Tables 2-4). Delabesse et al. [23] Ding et al. [24] Gil et al. [25] Rabkin et al. [26] Rabkin et al. [27] Year (

Conclusions
Represents a diploid, probably reactive, cell proliferation, which progressively increases the expression of antiapoptotic factors (cellular and viral).
Corroborates previous suggestions that KS could represent a reactive process, rather than a clonal proliferation.
Suggest a low level of DNA aneuploidy, but flow cytometry does not solve the dilemma of whether KS is a hyperplastic or neoplastic process. Support the view that KS (in males) develops into a clonal tumour yet initially is a hyperplastic reactive cell proliferation. Descriptions of clinical and morphological (histopathological) descriptions, such as plaque, nodular and patch stage, were frequently incomplete but still showed an important diversity of case combinations when studying the different outcomes, as follows:

2.
Clinical and morphological description of chromosomal aberrations studies: 58 (27%) of the 214 cases were HIV associated, 147 (69%) classic and 9 (4%) iatrogenic KS presentations. Only 3 studies [36,37,39] reported on morphological stages of these cases summing 3 (2%) plaque, 37 (17%) nodular, 23 (11%) lesion samples and 151 (70%) cases where it was not reported. Outcomes assessing the clonal nature of KS provided by the 20 included studies (Tables 2-4) resulted very heterogeneous (Figure 2). For a more comprehensive synthesis of the results, we decided to report retrieved outcomes grouped into the mentioned three categories of studied genetic alterations. However, differences in study aims, outcome definitions, applied methods and reported outcome measures limited seriously our possibility to pool and/or compare the published data. This heterogeneity in reported data was also the reason for ruling out a quantitative synthesis of the retrieved results, and the performance of a meta-analysis was excluded.

3.
Determining clonality: outcomes on clonality in KS samples were mostly determined by monoclonal patterns of gene inactivation or methylation and obtained mixed results. Four case series [24][25][26][27] obtained results suggesting that KS is a clonal neoplasm, while one study obtained results compatible with the description of a polyclonal cell [23]. All studies presented a high risk of bias as assessed by the adapted Quadas-2 tool due to bias inherent to their study design and insufficient reporting.

4.
Detecting mutations: Ten studies aimed to detect mutations in KS lesions, focusing three on p53 mutations [21,34,35] and seven on other different single mutations (IL-6, c-myc, LEC and BEC gene signatures, FGF3, HLA, FGF4 and KRAS). Each study applied different laboratory technics, determined different outcomes and reported diverse findings. Of these ten studies, three [21,29,32] reported negative results for the investigated mutation, and the other seven obtained outcomes suggested a possible relationship. Of the three studies [21,34,35] focusing on a possible role of p53 mutation in the KS oncogenesis, two [29,35] obtained results that point to a possible implication, and one case control study [21] with a large sample and well-performed statistical analysis failed in obtaining evidence of a possible association. These studies presented a high risk of bias, including the prospective case series [22] and the case control study [21] due to difficulties in assessing the internal and external validity of the studies based on the reported data.

5.
Demonstrating chromosomal aberrations: Finally, of the five selected studies [36][37][38][39][40] that investigated chromosomal aberrations, two studies [36,38] reported diploidy Reizis et al. [38] (a type of pattern for iatrogenic forms of KS) and aneuploid patterns for the classic form of KS. Another study [40] detected low levels of DNA aneuploidy, and the other two [37,41] reported results compatible with a hyperplastic reactive cell proliferation. Again, a high risk of bias was detected for all included studies due to limitations inherent to the study design and through a lack of detailed reporting of methods. Tables 2-4 summarize our findings. 5. Demonstrating chromosomal aberrations: Finally, of the five selected studies [36][37][38][39][40] that investigated chromosomal aberrations, two studies [36,38] reported diploidy Reizis et al. [38] (a type of pattern for iatrogenic forms of KS) and aneuploid patterns for the classic form of KS. Another study [40] detected low levels of DNA aneuploidy, and the other two [37,41] reported results compatible with a hyperplastic reactive cell proliferation. Again, a high risk of bias was detected for all included studies due to limitations inherent to the study design and through a lack of detailed reporting of methods. Tables 2-4 summarize our findings.

Discussion
We performed a systematic review of the peer-reviewed and published scientific literature on genetic alterations assessing the clonal nature of KS. The limited number of retrieved studies and the low-level of evidence of the retrieved studies [42,43], together with the low number of recent publications investigating this topic suggests that this potentially controversial topic has not been studied in depth from a broad, multidisciplinary perspective. Scientific interest in recent years has focused mainly on describing the viral oncogenic role through mechanistic studies [44][45][46][47][48] and relatively little attention has been paid to the analysis of factors influencing clonal growth in KS. However, the identification of such underlying mechanisms and subsequent definitions could lead to improvements in the clinical management of KS patients.
That said, there are several aspects of the pathogenesis of KS that are currently under investigation to determine the relevance of latency-associated nuclear antigen (LANA) and other HHV8 proteins in subverting normal endothelial cell biology to induce proliferation [49,50] and to describe the miRNA-34A effect [51]. Evidence from basic research proposing that HHV8 is an integrating oncovirus that causes amplification and activation of oncogenes needs to be further investigated by the sequencing of early and advanced lesions considering the latent state in which HHV8 has been reported to exist [52] as the clonality of HHV8 integration is sometimes questioned. While many studies have been published reporting genetic alterations in KS samples, few have analysed whether these are driver mutations that might be involved in oncogenesis [53,54].

Discussion
We performed a systematic review of the peer-reviewed and published scientific literature on genetic alterations assessing the clonal nature of KS. The limited number of retrieved studies and the low-level of evidence of the retrieved studies [42,43], together with the low number of recent publications investigating this topic suggests that this potentially controversial topic has not been studied in depth from a broad, multidisciplinary perspective. Scientific interest in recent years has focused mainly on describing the viral oncogenic role through mechanistic studies [44][45][46][47][48] and relatively little attention has been paid to the analysis of factors influencing clonal growth in KS. However, the identification of such underlying mechanisms and subsequent definitions could lead to improvements in the clinical management of KS patients.
That said, there are several aspects of the pathogenesis of KS that are currently under investigation to determine the relevance of latency-associated nuclear antigen (LANA) and other HHV8 proteins in subverting normal endothelial cell biology to induce proliferation [49,50] and to describe the miRNA-34A effect [51]. Evidence from basic research proposing that HHV8 is an integrating oncovirus that causes amplification and activation of oncogenes needs to be further investigated by the sequencing of early and advanced lesions considering the latent state in which HHV8 has been reported to exist [52] as the clonality of HHV8 integration is sometimes questioned. While many studies have been published reporting genetic alterations in KS samples, few have analysed whether these are driver mutations that might be involved in oncogenesis [53,54].
We retrieved only two studies with an appropriate study design for the investigation of such associations; however, both focused on epidemiological aspects of the disease and did not provide effect measures analysing the relationship of a mutation or clonality with KS. Nicolaides et al. [28] showed KRAS mutation of amplification in substantial numbers of advanced cases in which codons 13, 15, 16, 18 and 31 were implicated. A small number of patients with the TP53 mutation have been found and may be associated with progression [34,35]. TP53 immunohistochemistry has been used, and expression appears to increase with progression [55]. The ability of NGS to examine intra-tumoral heterogeneity, mutation load, copy number variation and temporal heterogeneity makes this a major gap in the current knowledge of KS.
Clonality of the virus can be shown [56,57] but is not direct evidence of clonality of the host cells. Indeed, this may simply represent clonal restriction following polyclonal infection [23]. However, in a large study of 98 patients, Duprez et al. [58] used an HHV-8fused terminal repeat (TR) to follow up an earlier study [59] to show that most lesions were oligoclonal and concluded that KS was a reactive process in most patients, although many samples were not informative [58].
They found that differences 'strongly suggest that disseminated lesions represent multiple distinct primary expansions of HHV-8-infected spindle cells originating from different infectious events rather than metastatic proliferations'. It is known that EBVinduced lymphoproliferative disease starts as an oligoclonal proliferation and can lead to monoclonal diffuse large B-cell lymphoma with the adoption of additional genetic mutations; however, the same has not been shown for KS-although it remains a possibility.
The chronic and slow evolution of classic-KS lesions, the partial reversibility of iatrogenic-KS lesions after diminution of immunosuppressive regimens and the complete epidemic-KS regression after anti-retroviral therapy seem more consistent with reactive proliferation [23,26,38,40]. Although there are occasional reports of spontaneous regression of individual lesions (and rarely of all disease) [60]. Dependence on cytokines, such as basic fibroblast growth factor, oncostatin M and interleukin 6, has been shown for the growth of Kaposi's sarcoma-derived cells in vitro [31]. However, a commonly used cell line (KS Y-1) derived from KS lesions does produce tumours in nude mice [61].
Detailed, and ideally longitudinal, NGS studies with examination of multiples of a range of early (cutaneous patch stage) and late (visceral) lesions in individual patients could provide evidence of clonal selection and progression. Such an approach would evaluate for neoplastic transformation of early lesions.

Conclusions
In conclusion, insufficient scientific evidence exists to describe the nature of KS. Current knowledge is insufficient to determine whether KS is a clonal neoplasm, or simply an aggressive reactive virus-driven lesion. Although evidence from mechanistic studies that points towards a reactive virus-driven process that gives rise to a clonal neoplasm is mounting, direct evidence for the pathogenic pathway would allow an evidence-based assessment of KS neoplastic status with all the implications that this may have for clinical practice and patient care. Its current neoplastic classification as a sarcoma is questionable, and regarding it as a 'Kaposi tumour' may be more accurate. Funding: This research received no external funding. The WHO Classification of Tumours is published by IARC on a not-for-profit basis, funded solely by sales of its books and subscription website.