Cancer of Unknown Primary ( CUP ): genetic evidence for a novel nosological entity? A case report
2020; Springer Nature; Volume: 12; Issue: 7 Linguagem: Inglês
10.15252/emmm.201911756
ISSN1757-4684
AutoresSilvia Benvenuti, Melissa Milan, Elena Geuna, Alberto Pisacane, Rebecca Senetta, Gennaro Gambardella, Giulia Maria Stella, Filippo Montemurro, Anna Sapino, Carla Boccaccio, Paolo M. Comoglio,
Tópico(s)Oral and Maxillofacial Pathology
ResumoReport8 June 2020Open Access Transparent process Cancer of Unknown Primary (CUP): genetic evidence for a novel nosological entity? A case report Silvia Benvenuti Silvia Benvenuti Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Melissa Milan Melissa Milan Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Elena Geuna Elena Geuna Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Alberto Pisacane Alberto Pisacane Pathology Unit, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Rebecca Senetta Rebecca Senetta Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Gennaro Gambardella Gennaro Gambardella Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (Naples), Italy University of Naples Federico II, Naples, Italy Search for more papers by this author Giulia M Stella Giulia M Stella Department of Medical Sciences and Infectious Diseases, Unit of Respiratory System Diseases, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy Search for more papers by this author Filippo Montemurro Filippo Montemurro Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Anna Sapino Anna Sapino Pathology Unit, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Carla Boccaccio Carla Boccaccio orcid.org/0000-0003-2620-9083 Laboratory of Cancer Stem Cells, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Department of Oncology, University of Turin Medical School, Candiolo (Turin), Italy Search for more papers by this author Paolo M Comoglio Corresponding Author Paolo M Comoglio [email protected] orcid.org/0000-0002-7056-5328 Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Silvia Benvenuti Silvia Benvenuti Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Melissa Milan Melissa Milan Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Elena Geuna Elena Geuna Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Alberto Pisacane Alberto Pisacane Pathology Unit, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Rebecca Senetta Rebecca Senetta Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Gennaro Gambardella Gennaro Gambardella Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (Naples), Italy University of Naples Federico II, Naples, Italy Search for more papers by this author Giulia M Stella Giulia M Stella Department of Medical Sciences and Infectious Diseases, Unit of Respiratory System Diseases, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy Search for more papers by this author Filippo Montemurro Filippo Montemurro Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Anna Sapino Anna Sapino Pathology Unit, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Department of Medical Sciences, University of Turin, Turin, Italy Search for more papers by this author Carla Boccaccio Carla Boccaccio orcid.org/0000-0003-2620-9083 Laboratory of Cancer Stem Cells, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Department of Oncology, University of Turin Medical School, Candiolo (Turin), Italy Search for more papers by this author Paolo M Comoglio Corresponding Author Paolo M Comoglio [email protected] orcid.org/0000-0002-7056-5328 Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy Search for more papers by this author Author Information Silvia Benvenuti1, Melissa Milan1, Elena Geuna2, Alberto Pisacane3, Rebecca Senetta4, Gennaro Gambardella5,6, Giulia M Stella7, Filippo Montemurro2, Anna Sapino3,4, Carla Boccaccio8,9 and Paolo M Comoglio *,1 1Molecular Therapeutics and Exploratory Research Laboratory, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy 2Oncology Outpatient Clinic, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy 3Pathology Unit, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy 4Department of Medical Sciences, University of Turin, Turin, Italy 5Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli (Naples), Italy 6University of Naples Federico II, Naples, Italy 7Department of Medical Sciences and Infectious Diseases, Unit of Respiratory System Diseases, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy 8Laboratory of Cancer Stem Cells, Candiolo Cancer Institute, FPO – IRCCS, Candiolo (Turin), Italy 9Department of Oncology, University of Turin Medical School, Candiolo (Turin), Italy *Corresponding author. Tel: +39 0119 93601; E-mail: [email protected] EMBO Mol Med (2020)12:e11756https://doi.org/10.15252/emmm.201911756 See also: V Davalos & M Esteller (July 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cancer of unknown primary (CUP) is an obscure disease characterized by multiple metastases in the absence of a primary tumor. No consensus has been reached whether CUPs are simply generated from cancers that cannot be detected or whether they are the manifestation of a still unknown nosological entity. Here, we report the complete expression and genetic analysis of multiple synchronous metastases harvested at warm autopsy of a patient with CUP. The expression profiles were remarkably similar and astonishingly singular. The whole exome analysis yielded a high number of mutations present in all metastases (fully shared), additional mutations (partially shared) accumulated one after another in a series, and few private mutations were unique to each metastasis. Surprisingly, the phylogenetic trajectory linking CUP metastases was atypical, depicting a common “stream”, sprouting a series of linear “brooks”, at variance from the extensive branched evolution observed in metastases from most cancers of known origin. The distinctive genetic and evolutionary features depicted suggest that CUP is a novel nosological entity. Synopsis Cancer of unknown primary (CUP) is a heterogeneous clinical syndrome characterized by metastases without primary tumor, an elusive biology and lack of effective treatments. We describe the genetic and evolutionary features of 15 synchronous and spatially distinct metastases from a CUP patient. CUP metastases were mostly genetically homogeneous, each one accruing an independent smaller mutational burden. Reconstruction of phylogenetic relationships among metastases suggested the presence of a common ancestor that linearly and rapidly accumulated mutations, and disseminated a progeny at subsequent stages of its evolution. The genomic and transcriptomic traits of CUP metastases prompted recognition of a distinct nosological entity. On the clinical ground, the unexpected genetic similarity among different CUP metastases, fosters a therapeutic strategy aimed at simultaneous eradication of the multiple lesions. The paper explained Problem Cancer of unknown primary is an obscure disease characterized by multiple metastases in the absence of a clinically detectable primary tumor. The elusive CUP biology results in the lack of pathogenesis-based therapy. Results Fifteen synchronous metastases from a single CUP patient were analyzed by whole exome and RNA sequencing and their phylogenetic tree was reconstructed. Surprisingly, a high percentage of mutations, including those in putative driver genes, were fully shared. Additional mutations accumulated one after the other in a series, and a few private mutations were unique to each metastasis. The phylogenetic trajectory linking CUP metastases depicted an evolution pattern reminiscent of a galaxy: a common “stream” sprouting a series of linear “brooks”. Impact The distinctive genetic and evolutionary features of CUPs suggest a biology different from metastases of cancers of known origin. On the clinical ground, the unexpected genetic similarity among different CUP metastases leaves room for a therapeutic strategy aimed at the simultaneous eradication of multiple lesions. Introduction In spite of representing 3–5% of all new cancer diagnoses, cancer of unknown primary (CUP) is the fourth most common cause of cancer-related deaths worldwide (Pavlidis & Pentheroudakis, 2012; Varadhachary & Raber, 2014) and remains a mysterious nosological entity sharing common traits: (i) early dissemination; (ii) unpredictable organ distribution; (iii) lack of tissue-specific differentiation markers; and (iv) poor prognosis. The elusive CUP biology results in the lack of effective, pathogenesis-based therapy (Golfinopoulos et al, 2009). The standard of care for CUPs is based on chemotherapy, driven by an empirical, semi-agnostic, approach based on histological suggestions from a panel of immunohistochemical markers (Fizazi et al, 2015). Until recently, major efforts have been directed to predict the tissue of origin by means of immunohistochemistry (Greco et al, 2012), gene expression (Hainsworth et al, 2013), miRNA (Søkilde et al, 2014), or epigenetic (Moran et al, 2016) profiling, with the assumption that the knowledge of the putative tissue of origin could dictate therapeutic strategies. Yet, a recently published clinical trial showed no advantages of a molecularly defined, site-specific chemotherapy regimen compared with an empirically chosen chemotherapy (Hayashi et al, 2019). Another approach, which awaits confirmation, consists of finding druggable molecular target(s) (Ross et al, 2014). Thus, mutational profiles could be useful to reveal CUPs’ vulnerabilities (the “precision medicine” approach). Genomic surveys of CUPs, performed on panels of selected cancer genes, have been recently presented (Ross et al, 2014; Löffler et al, 2016; Varghese et al, 2017; Zehir et al, 2017), but a distinguishable and specific genetic signature has not emerged and no actionable targets have been identified. Moreover, the typical multimetastatic presentation of CUPs might represent a further major challenge for precision medicine since the genetic makeup of each metastasis might be rather heterogeneous, undermining the outcome of therapies tailored on genetic alterations detected in the single lesion subjected to biopsy. On the theoretical ground, it remains an open question whether CUPs are a jumble of metastatic cancers where the primary cannot be detected, or they are a still unrecognized cancer type propelled by distinctive genetic and molecular features (Pentheroudakis et al, 2007). To answer, we tried to decipher the evolutionary trajectories linking the multiple and synchronous metastases arising in a patient with CUP, thus providing genetic evidence for a new nosological entity and hints to envisage targeted therapeutic interventions. Results A thorough diagnosis of CUP We studied in depth a 49-year-old male presenting with rapidly progressing multiple metastases in different sites. A thorough multistep workout was conducted following the ESMO guidelines, which excluded the presence of a primary tumor (Fizazi et al, 2015) (Fig 1A–D and Table EV1). Histology of an ultrasound-guided core biopsy of a breast metastasis revealed a poorly differentiated tumor with adenocarcinoma features (Fig EV1). The tumor was intensively immunoreactive for cytokeratins 7, AE1/AE3, and BCA225, and focally positive for cytokeratin 20, whereas it was negative for the markers listed in Table EV1 (Fig EV1). Cancer-associated genetic alterations scrutinized by OncoCarta™ were undetectable (Table EV2). The patient was offered a treatment in a phase 2 trial, assessing nab-paclitaxel-based doublet as first-line therapy in CUPs (AGNOSTOS trial, no. 008-IRCC-10IIS-14). Nevertheless, he progressed rapidly and after two cycles he was withdrawn from chemotherapy. Three months later the patient succumbed and underwent a “warm” autopsy. Fifteen spatially distinct metastases encompassing eight different organs/tissues were harvested: left axillary lymph node (n = 1), abdominal subcutis (n = 1), right colic flexure (n = 1), liver (n = 4), kidney (n = 2), gluteal subcutis (n = 1), mediastinum (n = 1), right-side breast (n = 1), and lung (n = 3). All investigated sites showed the same histology (Fig 1D), superimposable to the diagnostic breast biopsy (Fig EV1). Figure 1. CUP diagnosis A. Multistep ad excludendum diagnostic workflow: diagnosis performed in accordance with ESMO guidelines (Fizazi et al, 2015), starting from clinical evaluation and proceeding with the sequential examinations represented in clockwise order. &Serum and immunohistochemistry markers are listed in Table EV1. $Cancer-associated genes are listed in Table EV2. GI: gastrointestinal. B, C. Metastases distribution: The fifteen metastases were retrieved at warm autopsy from 8 tissues/organs. Samples are numbered according to the sequence of harvest at autopsy. D. Histology: All metastatic lesions are composed of poorly differentiated cells with an epithelial “flavor”. The neoplastic population is mainly arranged in solid nests and sheets with focal rudimental gland formation. Scale bar: 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. IHC analysisNineteen IHC reactions performed on the breast core biopsy performed during the ad excludendum CUP diagnosis. H&E: hematoxylin and eosin. Scale bar: 50 μm. Download figure Download PowerPoint Cancer of unknown primary diagnosis was further confirmed at the transcriptional level. RNA-seq analysis of metastases from six sites (right colic flexure, liver, kidney, mediastinum, breast, and lung) yielded gene expression profiles that were similar among each other but did not match the profiles available in the TCGA dataset of any conventional primary tumor (i.e., tumors originated in a recognizable organ) or metastases from known primaries (Fig 2). Two primary ovarian cancers used as controls displayed expression profiles similar to that of ovarian cancers deposited in the TCGA dataset. The hierarchical clustering analysis, based on correlation distance, was performed starting from the median expression profiles of each primary cancer type or metastases calculated from data deposited in TCGA. All median expression profiles preserve the tissue-specific identity feature (i.e., each metastasis clusters close to its tumor of origin; Fig EV2B). The transcriptional profiles of CUP metastases were unrelated to any putative tissue of origin (unlike metastases originated from known primaries) and enlightened a distinct expression signature. Figure 2. Hierarchical clustering analysis of gene expression profiles of six CUP metastasesTriplicate samples of L_03, L_04A, L_07A, L_09, L_10, and L_12C are compared with the expression profiles deposited in the TCGA dataset of a spectrum of primary tumors or metastases (meta) from known origin. Two ovarian cancers analyzed in house (CTR_OV1 and CTR_OV2) were used as controls and have expression profiles matching the profiles displayed by the ovarian cancers listed in TCGA (purple box). The acronyms are as follow: ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Quantile normalization and hierarchical clustering TCGA transcriptional profiles were normalized using the normalize quantiles function of preprocessCore package in the R statistical environment v3.6. Each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles. The hierarchical clustering analysis of TCGA primary tumors and metastases from known primaries (performed using median expression profiles) shows that each metastasis clusters close to its tumor of origin. In grey primary tumors for which metastases profiles were not available. Download figure Download PowerPoint Genomic characterization The genomic DNA extracted from the fifteen metastases was analyzed by whole exome sequencing (WES) and compared with the patient's own peripheral blood mononuclear cell DNA. The average depth of coverage was about 100× (Fig EV3). Single nucleotide variants (SNVs) and small insertions and deletions (InDels) were called with Strelka2 (Kim et al, 2018) to identify somatic alterations (Dataset EV1). Click here to expand this figure. Figure EV3. Coverage details Percentage of reads retained at each filtering steps over the total number of aligned sequencing reads. On the x-axis are aligned samples. onT, on target. Percentage of targeted base pairs that were sequenced at different depth of coverage. On the x-axis are aligned samples. Download figure Download PowerPoint The presence of germline mutations in the eight genes responsible for the main hereditary human tumors (BRCA1, BRCA2, CDH1, CTNN1A, MLH1, MSH2, MSH6, and p53), suspected because of the patient's familial history of multiple cancers, was excluded. In the fifteen CUP metastases, the genetic analysis yielded a number of nonsynonymous SNVs/InDels ranging from 144 to 376 (Fig 3A) for a total of 748 unique changes. Mutation rate varied from 6.00 to 8.00 mutations per 106 bases, thus excluding that any metastases were hyper-mutated (Network, 2012). The predominant signature of C>A transversions observed was consistent with a smoking signature (Alexandrov et al, 2016). All samples were microsatellite stable (Boland & Goel, 2010). Figure 3. Genetic analysis of fifteen CUP metastases Somatic mutations distribution: SNVs and InDels displayed by each metastasis (listed in the y-axis) were detected by Strelka2 tool (Kim et al, 2018). Brown traits represent single mutations. The number of fully shared (red), partially shared (orange), or private (yellow) mutations is indicated in the horizontal bar below. Five cancer-associated genes (TP53, ARID2, NTRK1, SMARCA4, and ZFHX3) mutated in all metastases are highlighted. Genetic similarity among metastases: the heatmap has been drawn according to the Jaccard index. The similarity ranges from 58 to 82% among all pairs with the exception of L_08 due to the scarce number of mutations. Clonal composition analysis: representative nested view of metastasis L_01. The five sub-clones (represented in different colors) inferred by clustering similarities of the variant allele frequencies (VAFs) follow a linear pattern. Variant allele frequency of fully shared mutations present in copy neutral regions. The box plot represents the fluctuation of the VAF (in the y-axis) of single mutations (grey dots) in each metastasis; each box represents the upper and lower quartiles, while the central short black line within each box represents the median; whiskers indicate variability outside the upper and lower quartiles. Reconstruction of the phylogenetic tree linking the fifteen CUP metastases: the model was reconstructed by taking into account the maximum likelihood molecular evolutionary tree according to Phylip (Felsenstein, 2005) and the incremental number of mutations. “Brooks” lengths are proportional to the amount of “private” mutations. Download figure Download PowerPoint The mutations identified were classified as (i) fully shared, (ii) partially shared, or (iii) private. (i) We considered fully shared the mutations displayed by at least 80% of metastases (i.e., 12/15). This threshold was chosen since differences in coverage, at any given region, would potentially produce false positives (i.e., mutations present only in one or few metastases) or false negatives (i.e., the absence of a mutation in one or more samples, due to insufficient coverage). By these criteria, 276 mutations were fully shared. (ii) On top of this common mutational pattern, additional mutations accumulated incrementally in each metastasis. These mutations, added one after the other in different metastases, were defined as partially shared. (iii) Finally, a few private mutations (from 4 to 48) were unique to each metastasis (Fig 3A). Indeed, the genetic concordance of the fifteen metastases, measured as a function of Jaccard similarity, ascertains a low degree of inter-metastases heterogeneity as all metastasis (but lesion L_8) display a similarity ranging from 58 to 82% (Figs 3B and EV4). The exception of L_08 is due to the scarce number of mutations displayed; nevertheless, out of the 144 somatic SNVs observed, four were partially shared and only four private. All remaining mutations were fully shared with other metastases. Click here to expand this figure. Figure EV4. Genetic similarity among metastases is specificThe genetic similarity among fifteen CUP metastases was calculated using Jaccard index, as shown in Fig 3B. Here, data from WES of three unrelated metastases (CTR_UNR1, CTR_UNR2, and CRT_UNR3) were included. The genetic similarity among those controls and metastases of the CUP patient (measured as function of Jaccard index) was zero (white squares). Download figure Download PowerPoint The clonal composition of the fifteen metastases, performed by clustering the variant allele frequency according to the algorithms SciClone (Miller et al, 2014) and ClonEvol (Dang et al, 2017), varied from 1 to 8, mostly following a linear pattern of evolution (Fig 3C). Clones harboring the same mutations expanded at different rates in different metastases (Fig 3D). Although identification of the genes involved in CUP onset and progression is not the focus of this analysis, it is worth to mention a few mutations occurring in known tumor-associated genes. In fact, within the 276 fully shared mutations (Dataset EV1), we found genetic lesions in the oncogene NTRK1 and the tumor suppressors TP53, ARID2, SMARCA4, ZFHX3, all of which have been described in CUPs (Zehir et al, 2017). Phylogenetic tree reconstruction Exploiting the whole exome analysis of the multiple metastases harvested from the same patient, and taking into account both synonymous and nonsynonymous mutations (SNVs and InDels) occurring in each metastasis and their purity-corrected frequencies (Phylip tool; Felsenstein, 2005), we reconstructed the phylogenetic relationships. While previous studies on metastases disseminated by known primary tumors revealed branched patterns mostly modeled as trees (Gerlinger et al, 2012) or stars (Sottoriva et al, 2015), analysis of the patient with CUP identified a single common “stream”, sprouting a series of sequential linear “brooks”. Figure 3E shows the sequential alignment of the fifteen metastases, based on the phylogenetic tree inferred by Phylip implemented by private mutations observed in each metastasis (Fig EV5). This picture suggests an unusual phylogenetic evolution consistent with the existence of a common ancestor that continues to accumulate mutations in a linear fashion, releasing over time collateral branches (the different metastases), each of which accrues an independent smaller mutational burden. The common ancestor is obviously undetectable in the patient body and may not necessarily display the features of a conventional cancer stem cell that generates a primary tumor mass. Rather, this ancestor might release in the bud its evolving progeny, which would rapidly disseminate and form metastases in tissues where microenvironmental conditions favor settlement and local growth. Click here to expand this figure. Figure EV5. Phylogenetic tree reconstructionThe phylogenetic tree linking the fifteen metastases reconstructed by the Phylip tool (as described in Materials and Methods) is suggestive of an expansion pattern of a galaxy. Download figure Download PowerPoint Discussion The conventional approach to therapy of cancers of unknown primary relies in pushing the molecular characterization of the metastatic lesions to the limits, to bet on a putative tissue of origin, and to treat the patients as if they were affected by a highly metastatic cancer of that tissue (Hayashi et al, 2019). It is possible that by this approach more (or possibly all) currently defined CUPs will be re-classified in a “tissue-gnostic” way. As an alternative, CUPs might turn out to be a nosological entity with distinctive traits. Identification of the gene(s) and the molecules responsible for these traits could provide hints to understand the biology of early tumor dissemination, from one side, and to pinpoint new selective therapeutic targets from the other. We approached the problem by a comprehensive comparative analysis of the transcriptional profiles, the genetic traits, and the phylogenetic relationship among multiple synchronous and spatially distinct metastases in an exemplary case of CUP. Such information has never been reported. The first surprise was the expression profiles shared by CUP metastases which were, otherwise, dissimilar from the profiles displayed by the plethora of normal and tumor cells (including metastases) deposited in the TCGA dataset. The second unexpected finding was the high degree of similarity among the mutational makeup of different CUP metastases, unlike what commonly observed among metastases from tumors of known origin (Gerlinger et al, 2012; Sottoriva et al, 2015). This similarity is surprising since the precocious dissemination of the disease would suggest high inter-metastases heterogeneity according to the parallel progression model (Naxerova & Jain, 2015). Indeed, when metastases from known primary tumors disseminate early, they continue to evolve independently, giving rise to a wide genetic divergence. However, the high degree of homogeneity among CUP metastases is consistent with the rapid clinical evolution: after homing into multiple tissues, founder cells generate metastases leading patients to death in such short a time that only minimal divergent evolution can take place. The incremental accumulation of “partially shared” and the presence of few “private” mutations in individual metastases allowed drawing the phylogenetic tree. The inferred evolutionary pattern is unusual in metastases originated from known primary tumors. As described, the phylogenetic trajectory depicted a “stream-like” path from which a number of linear “brooks” originated. This pattern is surprisingly reminiscent of the expansion of a galaxy (Fig EV5). An alternative botanical metaphor recalls the olive tree terminal shoot. This model is consistent with the presence of a common cell of origin with stem-like features that after accumulating the common set of mutations, including all those affecting the putative driver genes, became fully malignant, and acquired the ability to relentlessly proliferate and disseminate its progeny. Such progeny likely underwent further accrual of the “partially shared” mutations and modest divergence, in the meantime spreading across the organism. At different metastatic sites, the founder cell(s) generated metastases where the accumulation of “private” mutations was minimal. Although the identification of possible metastatic drivers is beyond the scope of the paper, the mutational analysis enlightened a few candidate genes—shared by all metastases—and many possible gene combinations that may interfere with key signaling pathways controlling invasive growth (Comoglio et al, 2018). Among the mutations conserved in all metastatic sites, it is worth mentioning truncation of TP53, and critical amino acid substitutions in the transcription factor ZFHX3, in the receptor NTRK1, and in the chromatin remodeling proteins ARID2 an
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