News & Views Insights from the genetic and transcriptional characterization of a cancer of unknown primary (CUP) Veronica Davalos1 & Manel Esteller1,2,3,4,* Cancer of unknown primary (CUP) defines empirical chemotherapy to improve survival develop following a distinctive biological a heterogeneous group of metastatic have been centered on two different strate- pathway different to the canonical meta- tumors that lack an identifiable primary gies: traditional tissue-gnostic or tissue- static cascade remains to be explored tumor, despite a standardized diagnostic agnostic approaches. For the first, tumor- (Fig 1). work-up (Fizazi et al, 2015). CUPs are type classification is the key to treatment In an effort to improve our knowledge on characterized by an aggressive clinical allocation. Tissue specificity of gene expres- this enigmatic entity, Benvenuti et al (2020) course, unusual metastatic pattern, and sion and epigenetic profiles (Moran et al, describe in this issue of EMBO Molecular poor prognosis. Research in this field has 2016) has guided the development of tumor- Medicine the molecular characterization of been encouraged to unravel the complex- type classifiers for CUPs (Moran et al, multiple synchronous and spatially distinct ity of this enigmatic entity and improve 2017). Case reports, case series, and non- metastases from a CUP patient. A total of 15 clinical management and survival of CUP randomized prospective studies of CUPs metastases were retrieved at warm autopsy patients. In this issue of EMBO Molecular have indicated that tumor-type specific ther- from nine different tissues of a 49-year-old Medicine, Benvenuti et al (2020) describe apies lead to improved outcomes; however, male patient. Whole-exome sequencing the molecular characterization of multiple randomized controlled trials have not clearly (WES) and RNA sequencing (RNAseq) were synchronous and spatially distinct metas- demonstrated their superiority in compar- performed on 15 and 6 metastases, respec- tases from a CUP patient, shedding light ison with empirical chemotherapy (Fizazi tively. The comprehensive genomic and on the evolutionary dynamic and distinc- et al, 2019; Hayashi et al, 2019). transcriptomic data generated in this report tive features of CUP. Considering that the tissue could no offer a unique opportunity to study the longer be the focus in CUPs, tissue-agnostic mechanistic origin and evolution of CUPs. EMBO Mol Med (2020) 12: e12685 approaches have also gained force in the last WES analysis identified a total of 748 See also: Benvenuti et al (July 2020) years. Molecularly driven therapies have nonsynonymous mutations in the 15 metas- already transformed the treatment landscape tases, 276 of them were fully shared. Inter- of several tumors harboring actionable estingly, the genetic similarity among all D espite the advances in medical imag- mutations, independently of the tissue of metastases pairs ranged from 58 to 82%, ing and molecular approaches, the origin. Along these lines, the ongoing phase revealing a low degree of inter-metastases primary site cannot be identified at II, randomized clinical trial CUPISCO heterogeneity. A similar scenario has the time of diagnosis in 1–2% of cancers (NCT03498521) will compare the efficacy recently been described in treatment-naive (Rassy & Pavlidis, 2020). The existence of of molecularly guided therapy versus metastases from patients with tumors of CUP as a real entity is further supported by standard platinum-based chemotherapy in known primary, where 66% of the muta- cases where the primary tumor cannot be CUP cases. tions per patient were clonal (homogeneous) identified even after extensive diagnostic Although the lack of a primary tumor in among metastases (Reiter et al, 2019). work-up or exhaustive research of post- CUPs could be explained by limited sensitiv- Genetic homogeneity seems to be the rule mortem autopsy. ity of imaging technologies for detecting rather than the exception in both scenarios, Clinical management of CUP patients is a very small tumors, or the possibility that the leaving room for the identification of poten- challenge in the oncology field. Efforts to primary tumor regresses or remains tially druggable targets to simultaneously expand the therapeutic alternatives beyond dormant, the existence of metastases that eradicate multiple lesions. Moreover, 1 Josep Carreras Leukaemia Research Institute (IJC), Barcelona, Catalonia, Spain 2 Centro de Investigacion Biomedica en Red Cancer (CIBERONC), Madrid, Spain 3 Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain 4 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Catalonia, Spain *Corresponding author. E-mail: mesteller@carrerasresearch.org DOI 10.15252/emmm.202012685 | EMBO Mol Med (2020) 12: e12685 | Published online 17 June 2020 ª 2020 The Authors. Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine 12: e12685 | 2020 1 of 3 EMBO Molecular Medicine Veronica Davalos & Manel Esteller CUP A Cancer of Small, undetectable unknown primary primary tumor Lack of/ undetectable primary tumor Unpredictable B metastatic pattern Primary tumor regression Aggressive clinical course Poor prognosis C Deregulated/ pre-malignant stem (like) cell © EMBO METASTATIC DISSEMINATION Figure 1. Hypothesized origins of CUP. (A) Small primary tumors undetectable with available imaging technologies; (B) primary tumors that regress or remain dormant; (C) metastases that develop following a distinctive biological pathway, different to the canonical metastatic cascade of dissemination from a primary tumor, perhaps from cells with stem cell attributes. considering the aggressive nature and rapid methodological limitations related to sample case of CUPs, stem cell attributes such as clinical course of CUPs, the presence of type (bulk sequencing) and depth of cover- migratory ability, plasticity, and high strong clonal oncogenic driver mutations age (< 50× in about 60% of targeted base renewal capacity could explain metastatic could also explain the lack of heterogeneity pairs) could diminish the accuracy of their dissemination without the requirement of in the CUP metastases. inferences, the authors suggest that the establishing a primary tumor. CUP metas- Genomic data of several metastases from phylogenetic tree reconstruction of CUP tases might originate from stem cells or non- the same CUP patient are also an optimal metastases is consistent with the presence of stem cells that have acquired these biologi- setting to infer tumor evolutionary dynam- a common cell of origin with stem-like cal properties due to DNA alterations, and ics. Since the complexity of cancer arises features that disseminate its progeny, an are able to settle and colonize different from evolutionary processes of mutations, attractive hypothesis to explain CUP origin organs. The phenotypic plasticity and undif- genetic drift, and selection, use of genomic that merits further investigation. ferentiated state of these cells could also be sequencing is an invaluable tool to under- The existence of cancer stem cells, first behind the unpredictable metastatic patterns stand temporal and spatial patterns of described in hematopoietic tumors and later observed in CUPs, in contrast to the metasta- somatic evolution (Turajlic et al, 2019). in several solid tumors, has also been sis organotropism of cancers of known Although in Benvenuti et al (2020) proposed as a driver of metastasis. In the primary. 2 of 3 EMBO Molecular Medicine 12: e12685 | 2020 ª 2020 The Authors Veronica Davalos & Manel Esteller EMBO Molecular Medicine An additional layer of information, the CUPs. With this aim, the TCGA program, the cisplatin and gemcitabine or systemic transcriptomes of six CUP metastases, has major effort to catalog the molecular alter- treatment tailored by molecular gene also been analyzed by Benvenuti et al ations in cancer, has launched a multiomic expression analysis in patients with carcinomas (2020). To our knowledge, this is the most project under our coordination to character- of an unknown primary (CUP) site (GEFCAPI comprehensive series of metastases from a ize CUPs encompassing genomic, transcrip- 04) [abstract LBA15_PR]. Ann Oncol 30(Suppl. 5): case of CUP reported in the literature and is tomic, and epigenomic data. This project v851 – v934 per se a valuable contribution to scientific aims at expanding our knowledge on the Hayashi H, Kurata T, Takiguchi Y, Arai M, Takeda knowledge. molecular mechanisms governing CUP K, Akiyoshi K, Matsumoto K, Onoe T, Mukai H, Transcriptomic analysis of the CUP carcinogenesis and will also be key to assess Matsubara N et al (2019) Randomized phase II metastases revealed a lack of correlation the existence of different CUP entities, and trial comparing site-specific treatment based with The Cancer Genome Atlas (TCGA) to identify potential therapeutic targets that on gene expression profiling with carboplatin datasets of 33 different tumor types, a can be translated to clinical practice. Finally, and paclitaxel for patients with cancer of finding certainly unexpected. Although the increasing examples of successful use of unknown primary site. J Clin Oncol 37: 570 – 579 unique features of CUPs could arise from a immunotherapies in several tumor types Moran S, Martínez-Cardús A, Sayols S, Musulén distinctive transcriptome, this conclusion encourages the scientific community to E, Balañá C, Estival-Gonzalez A, Moutinho C, contrasts with the achievement of CUP clas- explore this approach to improve the dismal Heyn H, Diaz-Lagares A, de Moura MC et al sifiers based on tissue-specific expression outcome of CUP patients. (2016) Epigenetic profiling to classify cancer profiles (Moran et al, 2017). Further studies of unknown primary: a multicentre, to support this finding are encouraged, as Conflict of interest retrospective analysis. Lancet Oncol 17: well as inclusion of series of stem cells/ ME is a consultant of Ferrer International and 1386 – 1395 stem-like cells in the correlation analysis. Quimatryx. Moran S, Martinez-Cardús A, Boussios S, Esteller M Besides, additional caution should be taken (2017) Precision medicine based on considering that CUP samples analyzed in References epigenomics: the paradigm of carcinoma of this study derived from warm autopsy. Benvenuti S, Milan M, Geuna E, Pisacane A, unknown primary. Nat Rev Clin Oncol 14: Research autopsy approach holds a Senetta R, Gambardella G, Stella GM, 682 – 694 tremendous potential to contribute to under- Montemurro F, Sapino A, Boccaccio C et al Rassy E, Pavlidis N (2020) Progress in refining stand physiological and pathological condi- (2020) Cancer of Unknown Primary 1 (CUP): the clinical management of cancer of tions. However, even over relatively short genetic evidence for a novel nosological unknown primary in the molecular era. Nat timeframes, death introduces a bias in cellu- entity? A case report. EMBO Mol Med 12: Rev Clin Oncol https://doi.org/10.1038/s41571- lar transcriptomes. Using mRNA sequencing e11756 020-0359-1 data from multiple tissues of post-mortem Ferreira PG, Muñoz-Aguirre M, Reverter F, Sá Reiter JG, Baretti M, Gerold JM, Makohon-Moore donors obtained from the Genotype Tissue Godinho CP, Sousa A, Amadoz A, Sodaei R, AP, Daud A, Iacobuzio-Donahue CA, Azad NS, Expression (GTEx) database, a recent Hidalgo MR, Pervouchine D, Carbonell- Kinzler KW, Nowak MA, Vogelstein B (2019) An systematic study found that the post-mortem Caballero J et al (2018) The effects of death analysis of genetic heterogeneity in untreated interval (PMI) impacts RNA stability in a and post-mortem cold ischemia on human cancers. Nat Rev Cancer 19: 639 – 650 very tissue-dependent manner (Ferreira tissue transcriptomes. Nat Commun 9: 490 Turajlic S, Sottoriva A, Graham T, Swanton C et al, 2018). The comparison of transcrip- Fizazi K, Greco FA, Pavlidis N, Daugaard G, Oien K, (2019) Resolving genetic heterogeneity in tomes of TCGA normal tissue counterparts Pentheroudakis G (2015) ESMO Guidelines cancer. Nat Rev Genet 20: 404 – 416 with normal tissues obtained at warm Committee. Cancers of unknown primary site: autopsy of the same CUP patient could ESMO Clinical Practice Guidelines for diagnosis, further support the unique transcriptomic treatment and follow-up. Ann Oncol Suppl 5: profile of the CUP metastases reported by v133 – v138 License: This is an open access article under the the authors. Fizazi K, Maillard A, Penel N, Baciarello G, terms of the Creative Commons Attribution 4.0 Multicentric, multidisciplinary, and Allouache D, Daugaard G, Van de Wouw A, License, which permits use, distribution and repro- multiomics studies are needed to decipher Soler G, Vauleon E, Chaigneau L et al (2019) A duction in any medium, provided the original work the biological and clinical complexity of phase III trial of empiric chemotherapy with is properly cited. ª 2020 The Authors EMBO Molecular Medicine 12: e12685 | 2020 3 of 3