Cancer Metastasis Rev DOI 10.1007/s10555-016-9611-7

Genetic traits for hematogeneous tumor cell dissemination in cancer patients Simon A. Joosse 1 & Klaus Pantel 1

# Springer Science+Business Media New York 2016

Abstract Metastatic relapse in patients with solid tumors is the consequence of cancer cells that disseminated to distant sites, adapted to the new microenvironment, and escaped systemic adjuvant therapy. There is increasing evidence that hematogeneous dissemination starts at an early stage of cancer progression with single tumor cells or cell clusters leaving the primary site and entering the blood circulation. These circulating tumor cells (CTCs) can extravasate into secondary tissues where they become disseminated tumor cells (DTCs). Patients might relapse years after initial resection of the primary tumor when DTCs become overt metastases. Current diagnostic strategies for stratification of therapies against metastatic cells focus on the primary tumor tissue. This approach is based on the availability of stored primary tumors obtained at primary surgery, but it ignores that the DTCs might have evolved over years, which can affect the antimetastatic drug response. However, taking biopsies from metastatic tissues is an invasive procedure, and multiple metastases located at different sites in an individual patient show marked genomic heterogeneity. Thus, capturing CTCs from the peripheral blood as a Bliquid biopsy^ has obvious advantages in particular when repeated sampling is required for monitoring therapies in cancer patients. However, the biology behind tumor cell dissemination and its contribution to metastatic progression in cancer patients is still subject to controversial

* Simon A. Joosse [email protected] Klaus Pantel [email protected]

1

Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany

discussions. This manuscript reviews current theories on the genetic traits behind the spread of CTCs and progression of DTCs into overt metastases. Keywords Cancer . Circulating tumor cells . Disseminated tumor cells . Metastasis . Genetic progression

1 Introduction The detection of circulating tumor cells (CTCs) in blood is a challenging exercise because of their rarity among the enormous amount of normal cells (about 1 in 10,000,000) [1–3]. Combinations of tumor cell-specific enrichment and highly sensitive detection methods can make the identification of these cells possible [4], and automated systems allow for routine diagnostics [5]. Despite the well-accepted concept of metastatic inefficiency, numerous studies in diverse tumor entities have shown that enumeration of CTCs in blood of cancer patients is significantly correlated with metastatic relapse and reduced overall survival [6]. Further characterization of CTCs for the presence of therapeutic targets (e.g., expression of ER or ERBB2), resistance-conferring gene mutations (e.g., KRAS mutations), or expression of splice variants of target receptors (e.g., androgen receptor) may yield potential predictive value, enabling tailored therapy in the near future [7–9]. CTCs have separated from the primary tumor mass and entered the systemic blood circulation by intravasation. Separation from the primary tumor may happen via active or passive routes [2]. Active dissemination can result in individual cell spread or collective migration through the transformation of epithelial cells to a more mesenchymal phenotype [10]. Passive dissemination may occur by cell clusters breaking off from the tumor mass into the circulation. Extravasation happens when CTCs adhere to the vascular wall and leave the

Cancer Metastasis Rev

systemic circulation at a distant site [11]. Once metastasis has manifested, new CTCs may find their way into the circulation that can lead to a further increase of the body’s total tumor load at other distant sites. Current treatment strategies directed against metastatic disease are frequently based on the histological and molecular information from the primary tumor [12]. However, in many cancer entities such as breast cancer, the latency between primary diagnosis and clinical evidence of metastasis may be more than a decade. The period between primary diagnosis and metastatic relapse provides ample time for natural and therapy-induced selective pressure and genetic evolution of the residual tumor cells. Adjuvant therapy and/or changes in the host (e.g., hormone or immune status) can alter the spectrum of cancer clones in an individual patient. Thus, reanalysis of cancer cells at the time of metastatic relapse seems to be important to tailor antimetastatic therapies [13]. Tissue biopsies may provide the means for the clinicians to estimate the relapse’s response to therapy; however, the invasive and burdensome procedure might be prohibited by the locations of the metastases. Furthermore, the dynamics in treatment response requires repetitive histological and molecular screening. Hence, peripheral blood would be an easier alternative source to obtain real-time information on the actual status of cancer cells. Monitoring and characterization of CTCs can be employed as surrogate Bliquid biopsy^ to probe the genetic landscape of metastatic cells located at different distant sites, which might provide companion diagnostics leading to individualized treatment during genetic progression of advanced cancer. However, the biology behind hematogeneous tumor cell dissemination and its contribution to metastatic progression in cancer patients is still subject to controversial discussions. Here, we review the facts and theories about tumor cell dissemination in cancer patients and discuss the clinical implications for improving cancer staging and therapy. This manuscript provides no comprehensive review but rather describes some of the most controversial areas with a focus on genetic traits. We hope that this may complement the recent reviews on CTCs published by us and other groups [2, 14–17].

2 Dissemination and parallel progression of tumor cells It has been long disputed whether the initiation of metastasis is an early or late event in cancer evolution (Fig. 1a). Based not only on the latency between clinical symptoms of a primary tumor and metastatic disease but also on the infrequent occurrence of primary metastasis, genetic progression of cancer has long been considered a linear process of stepwise accumulation of genetic mutations and epigenetic changes with cancer metastasis at its final stage [18]. For colorectal cancer, it is well described that neoplastic cells may attain specific

A Genec progression

Primary tumor

Late disseminaon Circulang tumor cells (CTCs)

Early disseminaon

B

C

Metastasis in distant organ (e.g., bone)

Clonal progression

Clonal cooperaon

Fig. 1 Possible scenarios of genetic progression towards metastasis. During all stages of cancer genetic progression, tumor cells are released into the blood stream and circulating tumor cells will travel to distant organs (a). Early stage tumor cells might need to undergo genetic progression to become a metastasis (b), whereas late stage tumor cells have acquired all necessary mutations and may be able to grow independently or in clonal cooperation (c)

mutations and chromosomal aberration and develop into low-grade adenoma, followed by high-grade adenoma [19]. Accumulation of more DNA mutations will transform the colorectal tumor cells into invasive carcinoma that may eventually lead to liver or other distant metastases [19, 20]. Interestingly, epithelial cells in blood from patients with benign colon disease and early stage breast cancer [6, 21] have been detected, suggesting that tumor cell dissemination might already occur at an early stage in cancer development and disseminated tumor cells (DTCs) do not necessarily have to originate from a small subpopulation at a later stage [22]. Further evidences pointing into this direction are gene expression profiling studies that can provide good predictions for metastatic recurrence based on the expression profiles of

Cancer Metastasis Rev

primary breast tumors due to the high similarity between primary and metastatic disease [23, 24]. On the other hand, early tumor cell dissemination also provides the means for parallel progression of the primary and disseminated tumor cells into genetically different clones. Confirmation of this notion is the finding of 19 additional mutations in a case of metastatic breast cancer detected by whole genome sequencing, which were not present in the primary tumor diagnosed 9 years earlier [25]. One of the consequences parallel progression can have is failure of anticancer therapy in the metastatic setting if the treatment has been dictated by the molecular or histological characteristics of the primary tumor [7]. Additional evidence for early dissemination is provided by comparative analyses of copy number alterations between DTCs found in the bone marrow and primary tumors [26, 27]. Whereas the numerous chromosomal aberrations found in the primary tumor indicate genetically evolved cells, fewer copy number changes found in DTCs indicate dissemination at an early stage at which the tumor also did not exhibit many chromosomal aberrations yet. Mouse experiments have even shown the occurrence of early dissemination by atypical hyperplasia cells from breast and pancreas tissue [28, 29], but these findings are not yet confirmed in patients. Taken together, one of most important conclusions that can be made from these studies is that early disseminated cells are also able to survive in the systemic circulation and home to foreign tissues [30]. Nevertheless, the discussion involving whether metastasis initiation is an early or late event continues on whether the dissemination of premature cancer cells is clinically relevant, as their ability of becoming an overt metastasis is still unknown. The observed genetic similarities between metastases and primary tumors [23] appear to be in contrast with parallel progression nevertheless does not exclude it if one considers the following scenario. Immature tumor cells disseminated from an early stage primary tumor and undergo further genetic evolution to become an overt metastasis [31]. Although this process takes several years, a combination of similar selection pressures from within the host (e.g., immune surveillance), as well as the same cell of origin, could eventually result in similar genetic evolution of the metastasis and the primary tumor (Fig. 1b). Remarkable similarities of copy number changes can be found within the same breast cancer subtypes between different individuals, as a consequence of the cell of origin [32–34]. It would therefore not be surprising if the cell of origin would be one of the driving forces of making up both a primary tumor’s and metastasis’ genomic landscape within an individual [35, 36]. Thus, the primary and metastatic cells progress independently, but this progression results in a similar genomic landscape characteristic for each individual patient. Early dissemination and parallel progression might also explain the occurrence of primary metastasis in patients with cancer of unknown primary (CUP) where the primary tumor

might have spontaneously regressed before any clinical symptoms have arisen [37]. CUP occurs in about 5 % of all cancer cases and is therefore not so rare [38]. Nevertheless, CUP can also be explained without parallel progression. The different microenvironments of primary and metastatic sites can selectively favor outgrowth of genetically identical tumor cells at the metastatic site, while growth of these cells is abrogated at the primary site. Previous experimental studies support the concept that the microenvironment determines the outgrowth of tumor cells with the same genetic shape [39, 40]. Contradictory to parallel progression is the low proliferation rate of DTCs, as the expression of the proliferation marker Ki-67 in the bone marrow DTCs is reported to be a rare event [41]; therefore, the likelihood that these cells gain new genetic mutations during the dormancy period before they start to form overt metastases is limited. Consistent with these findings are the infrequent occurrence of tumor cell clusters in the bone marrow of breast cancer patients and DTCs in breast cancer being found primarily as single cells even years after primary diagnosis and treatment [42]. However, we cannot exclude that DTCs proliferate at other distant sites that may also contain more cell clusters. Unfortunately, almost nothing is known about DTCs in distant organs other than the bone marrow because of the lack of appropriate markers that distinguish epithelial tumor cells in an epithelial organ such as the liver or lungs. Thus, it remains unclear to which extent the DTC data obtained from bone marrow analyses can be extrapolated to DTCs in other organs. Each organ has a special tissue architecture which might influence the growth rate of DTCs, as already proposed by Paget in his famous Bseed and soil^ hypothesis more than 100 years ago [43]. For example, the bone marrow has niches for the hematopoietic stem cells that can be occupied by DTCs and may keep these cells in a dormant state [44]. The dormant state appears to protect DTCs from chemotherapy, as previously indicated in breast cancer patients [45]. Nevertheless, based on the current knowledge, we would like to propose an alternative scenario of cancer metastasis in relation to genetic progression. Tumor cells continuously disseminate from the primary lesion during cancer development until the tumor are being diagnosed and resected. Those cells that disseminated later have a higher chance of becoming an overt metastasis because the primary tumor was genetically more progressed than at earlier stages of development (Fig. 1c). This theory is supported by the observation that DTCs are an important prognostic indicator of metastatic relapse [46], while DTCs are also found in up to 20 % of the patients with ductal carcinoma in situ (DCIS) at primary diagnosis [28, 47], although the subsequent development of overt metastasis in these patients is rare (3 %). Furthermore, this concept would also explain the well-known clinical observation that the local size of the primary tumor (called T-stage) is correlated with metastatic relapse in all types of solid tumors.

Cancer Metastasis Rev

Thus, the development of the primary tumor is coupled to the risk of metastasis in most cancer patients. More support of our theory comes from the observation that the detection of DTCs with proliferative potential and many copy number alterations predict unfavorable prognosis in cancer patients [48]. Based on the chromosomal aberrations, these DTCs presumably should originate from late stage tumors. Taken together, these results indicate that genetic progression is not necessarily the driver behind dissemination per se since tumor cells start to disseminate very early during tumor development. Nonetheless, we cannot exclude that genetic progression affects the rate of dissemination. Taking into consideration that circulating tumor cells (CTCs) have a half-life expectancy of about 1 h [49] and DTCs are in general in a dormant state [30], it is not expected that these cells undergo any genetic progression during their time in systemic circulation or after dissemination in distant organs. It may, therefore, be concluded that late dissemination of genetically progressed tumor cells should have a much higher chance of generating metastatic outgrowth than Bgenetically immature^ cells in the beginning of tumor development.

3 Genetic heterogeneity among CTCs Intratumoral phenotypical heterogeneity, as defined by, e.g., protein expression, treatment response, and growth rate, has been widely accepted already for many decades [50]. Heterogeneity is caused by selection pressure from the tumor microenvironment, local or systemic treatment, and Darwinian forces. During the progression of cancer, tumor cells genetically evolve by accumulating DNA point mutations, epigenetic changes, translocations, copy number aberrations, and inversions. During this so-called clonal evolution, a natural positive selection is made for advantageous mutations and a negative selection for deleterious mutations. Although different evolution models are described to explain tumor progression, fact is that cancer is a genetically heterogeneous disease [51]. An example of genetic heterogeneity is given by a recent study from Neve et al. The authors investigated chromosomal aberrations in two breast tumors by performing shallow, whole-genome, next-generation sequencing of 100 single cells from each tumor [52]. Although a limited amount of genetically different clones could be identified based on copy number aberrations, interestingly, no intermediate steps in genetic progression were found. This might be the consequence of the distribution of cells in different stages and the respective limited amount of analyzed cells, or it might indicate a strong selection for genetic stages in which tumor cells can survive. In light of the genetic heterogeneity of primary tumors, it is expected that both DTCs and CTCs should also be genetically heterogeneous if we accept the hypothesis that they can potentially originate from any tumor clone within the primary

lesion. Thus, CTCs might be an excellent surrogate marker for the tumor’s genetic landscape to determine therapy options in a heterogeneous background. This liquid biopsy could be clinically relevant in patients where biopsies of the primary tumor or metastases are difficult to obtain, especially repeatedly over time. For example, the detection of amplification of epidermal growth factor receptor (EGFR) is therapeutically relevant for cancer management; however, de novo resistance may already be present in small subpopulations of the tumor easily missed by the routine diagnostics. Therefore, the characterization of single tumor cells for amplification of EGFR, as well as mutation detection in signaling pathway genes downstream of EGFR, could predict a negative drug response and further facilitate tailored therapy [53–55]. It is observed that genetically heterogeneous tumors are more aggressive, have a higher metastatic rate, and are more resilient against therapy [56–58]. This observation has been studied in a xenograft model where monoclonal tumors were compared with polyclonal tumors that were created by lentiviral overexpression of cancer-promoting factors in a breast cancer cell line [59]. The mixture of subclones led to larger and more metastatic tumors as compared with the monoclonal tumors having the same genetic background. Evidence is accumulating that ecological interaction between tumor subclones plays an important role in cancer survival, progression, and metastasis [50]. As a consequence of positive interactions, tumor progression is accelerated because tumor cells do not require to fully transform genetically and can benefit from each other. In independent studies using mouse models, it has been shown that upon engrafting two different cell populations, one cell type provided the other cells with the ability to metastasize, whereas the clones could not disseminate on their own or even suffered necrotic collapse [59, 60]. These results imply that genetic heterogeneity must be maintained in order for a tumor to survive. In line with this are the results we recently obtained while establishing a cell line derived from CTCs from a colorectal cancer patient [61]. In order to characterize this cell line, one single cell and two cell clusters were analyzed for copy number aberrations by nextgeneration sequencing. Although similarities between the cells were prominent, several differences of copy number aberrations of whole chromosome arms could also be discerned. These results imply that the CTC population was already genetically heterogeneous when obtained from the patient and remained this way during further proliferating, presumably in clonal cooperation. Ongoing experiments will now explore whether these subclones require positive interaction or may also be able to survive monoclonally. Further evidence for growth advantages of genetic heterogeneity was recently provided by Aceto and colleagues [62]. In their study, the authors showed that CTC clusters composed of cells from difference clones have an increased metastatic capacity of up to 50-fold as compared to single CTCs.

Cancer Metastasis Rev

Taken together, it may be speculated that early DTCs may lack the genetic heterogeneity and ecological interaction with genetically different subclones, which might limit their capacity to grow into a metastasis. Further genetic progression of the primary tumor will result in the release of more genetically different CTCs and CTC clusters into the circulation, leading to an increased possibility of metastatic outgrowth by positive interaction of complementary clones (Fig. 1c).

4 Clinical implication of genetic progression and transcriptional plasticity in metastasis One of the major issues clinicians are facing in the management of metastatic cancer patients is tumor heterogeneity. Phenotypic and genotypic heterogeneity will eventually lead to therapy resistance after selection pressure has taken place [63, 64]. Evidence is growing that mutations found in cancer recurrence and have directly been linked to be responsible for therapy resistance may already have been present in small subpopulations in the primary tumor. For example, a recent study by Heitzer et al. on metastatic colorectal cancer demonstrated unique driver mutations in CTCs when comparing with matched primary tumors and metastases. However, deep sequencing of the primary tumors revealed the presence of these mutations at subclonal level [65]. These small subclones are difficult to trace by routine diagnostics and missed by the DTC analyses (e.g., conventional CGH or LOH analyses) that have led to the parallel progression concept [26]. Only DTCs originating from therapy refractory subclones can grow out to a metastasis, while therapy-sensitive tumor cells will be eradicated. Thus, relapse of disease requires renewed diagnostics on such specific clones. However, undergoing needle biopsy of metastatic lesions in relapsed patients repeatedly for therapy monitoring is invasive and associated with considerable risks, especially in relation to the relapse’s location (e.g., brain or lung metastases) [66]. As Heitzer and colleagues demonstrated for colorectal cancer [65], using CTCs as real-time liquid biopsy can provide an alternative for routine, minimal-invasive diagnostics, which can be applied during the course of genetic progression of the malignancy. As discussed above, it is not expected that many CTCs and DTCs undergo genetic evolution based on their low proliferation rate. On the other hand, tumor cells may have to undergo transcriptional changes in order to metastasize, survive dissemination, and grow into secondary tumors. First of all, epithelial-mesenchymal transition (EMT), a reversible cellular dedifferentiation program, is hypothesized to be responsible for promoting motility of cancer cells. During this transition to a mesenchymal phenotype, epithelial tumor cells partially lose their apical-basal polarization and cell-cell contacts [67]. Yu and colleagues showed that CTCs originating from aggressive triple-negative and ERBB2-overexpressing breast tumors

expressed more mesenchymal-associated markers, suggesting an active dissemination, whereas CTCs from less aggressive luminal tumors expressed more epithelial-associated markers [68]. Once CTCs have left the blood circulation and have found a niche, further adaptation is required in order to survive in the foreign environment. DTCs isolated from prostate cancer patients’ bone marrow show a high resemblance on transcriptional level with their neighboring bone marrow cells, suggesting a cellular plastic response to the microenvironment [69]. In the last step of the metastatic cascade, DTCs will escape dormancy and initiate their proliferation program again [30], which might enable them to acquire new genetic alterations and pass them on to their daughter cells. Plasticity of tumor cells may also contribute to therapy failure. RNA profiling of single CTCs from castrationresistant metastatic prostate cancer patients not only revealed heterogeneity within individual subjects but also the expression of different androgen receptor splice variants within single CTCs [70]. This is in contrast to the more homogeneous AR signaling in CTCs of untreated prostate cancer patients [71] and may confer resistance to antiandrogen therapies. Thus, RNA analyses of CTCs can provide important predictive information for treatment responses and survival [9].

5 Conclusions Immunocytochemical detection of DTCs in bone marrow of early stage cancer patients (e.g., early stage invasive breast cancer or even DCIS) and subsequent single cell genetic analyses of these DTCs have indicated that tumor cells disseminate at an early stage of cancer development when only few chromosomal aberrations have manifested. However, the methods used for the genomic analyses are rather outdated, and the results need to be therefore revisited in light of the recent development of next-generation sequencing technologies. The low proliferation rate of DTCs and short half-life time of CTCs suggest little subsequent genetic progression at the single cell level after dissemination. These findings contradict the genetically progressed, complex profiles of overt metastases [72]. Therefore, we hypothesize that CTCs from a tumor at late stage have a higher chance of initiating metastatic outgrowth then those shed from tumors at early stage, which is consistent with the significant correlation between T-stage and prognosis in cancer patients. Furthermore, tumor heterogeneity, as a consequence of prolonged cancer development, may greatly add to the survival chances and metastatic capacity due to ecological interaction between the different subclones. Additional parallel progression might occur in the late exponential growth phase of DTCs towards the development of metastases, and this might modulate the risk to develop gross metastases but the

Cancer Metastasis Rev

magnitude of this effect is unclear. The close resemblance between the primary and metastatic lesions of the same patient argues against a fundamental role of parallel progression in metastatic development. Mouse models are helpful tools to study metastatic progression [30], but we should keep in mind that the life span of mice is approximately 2 years, which is much shorter than the time required for the development of gross metastases in cancer patients except for some very aggressive tumor entities such as pancreatic cancer. On the other hand, blood or bone marrow analyses in cancer patients provide just snapshots of the metastatic progression process, and in vivo tracing of single CTCs or DTCs in humans is not possible. More in-depth genetic characterization together with functional studies on CTCs and DTCs collected over the course of the disease of individual patients together with cross-validation of the findings in appropriate mouse models might help to unravel the mysteries of metastasis in cancer patients [30]. Repeated sampling of tumor cells is also required to predict and monitor therapy response. Where tissue needle biopsies might not be an option, CTCs from blood may function as liquid biopsy and can be employed in single cell genomic analyses. The validation of liquid biopsy assays is an important task of the new European consortium CANCER-ID which comprises more than 30 institutions from academia and industry (www.cancer-id.eu). This effort will hopefully lead to new reliable biomarker tools that can be used as companion diagnostics in clinical trials testing new drugs aimed to eradicate metastatic cells. Acknowledgments The authors receive support from CANCER-ID, an Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115749, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and EFPIA companies’ in kind contribution. This work was further supported by the European Research Council Advanced Investigator grant 269081 DISSECT (KP).

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

References 17. 1.

2.

3.

4.

Muller, V., Alix-Panabieres, C., & Pantel, K. (2010). Insights into minimal residual disease in cancer patients: implications for anticancer therapies. European Journal of Cancer, 46(7), 1189–1197. doi:10.1016/j.ejca.2010.02.038. Joosse, S.A., Gorges, T.M., & Pantel, K. (2014). Biology, detection, and clinical implications of circulating tumor cells. EMBO Molecular Medicine, 7(1),1–11. doi: 10.15252/emmm.201303698. Joosse, S. A., & Pantel, K. (2013). Biologic challenges in the detection of circulating tumor cells. Cancer Research, 73(1), 8–11. doi:10.1158/0008-5472.CAN-12-3422. Joosse, S. A., Hannemann, J., Spotter, J., Bauche, A., Andreas, A., Muller, V., et al. (2012). Changes in keratin expression during metastatic progression of breast cancer: impact on the detection of circulating tumor cells. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 18(4), 993–1003. doi:10.1158/1078-0432.CCR-11-2100.

18.

19.

20.

21.

Riethdorf, S., Fritsche, H., Muller, V., Rau, T., Schindlbeck, C., Rack, B., et al. (2007). Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 13(3), 920–928. doi:10.1158/1078-0432.CCR-06-1695. Zhang, L., Riethdorf, S., Wu, G., Wang, T., Yang, K., Peng, G., et al. (2012). Meta-analysis of the prognostic value of circulating tumor cells in breast cancer. Clinical Cancer Research An Official Journal Of the American Association for Cancer Research, 18(20), 5701–5710. doi:10.1158/1078-0432.CCR-12-1587. Babayan, A., Hannemann, J., Spotter, J., Muller, V., Pantel, K., & Joosse, S. A. (2013). Heterogeneity of estrogen receptor expression in circulating tumor cells from metastatic breast cancer patients. PLoS ONE, 8(9), e75038. doi:10.1371/journal.pone.0075038. Schramm, A., Friedl, T. W., Schochter, F., Scholz, C., de Gregorio, N., Huober, J., et al. (2015). Therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer: concept of the DETECT study program. Archives of Gynecology and Obstetrics. doi:10.1007/s00404-015-3879-7. Antonarakis, E. S., Lu, C., Wang, H., Luber, B., Nakazawa, M., Roeser, J. C., et al. (2014). AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. The New England Journal of Medicine, 371(11), 1028–1038. doi:10.1056/NEJMoa1315815. Thiery, J. P., Acloque, H., Huang, R. Y., & Nieto, M. A. (2009). Epithelial-mesenchymal transitions in development and disease. Cell, 139(5), 871–890. doi:10.1016/j.cell.2009.11.007. Lange, T., Samatov, T. R., Tonevitsky, A. G., & Schumacher, U. (2014). Importance of altered glycoprotein-bound N- and Oglycans for epithelial-to-mesenchymal transition and adhesion of cancer cells. Carbohydrate Research, 389, 39–45. doi:10.1016/j. carres.2014.01.010. Dancey, J. E., Bedard, P. L., Onetto, N., & Hudson, T. J. (2012). The genetic basis for cancer treatment decisions. Cell, 148(3), 409–420. doi:10.1016/j.cell.2012.01.014. Dienstmann, R., Rodon, J., & Tabernero, J. (2013). Biomarkerdriven patient selection for early clinical trials. Current Opinion in Oncology, 25(3), 305–312. doi:10.1097/CCO.0b013e32835ff3cb. Alix-Panabieres, C., & Pantel, K. (2014). Challenges in circulating tumour cell research. Nature Reviews Cancer, 14(9), 623–631. doi: 10.1038/nrc3820. Haber, D. A., & Velculescu, V. E. (2014). Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA. Cancer Discovery, 4(6), 650–661. doi:10.1158/2159-8290.CD-13-1014. Krebs, M. G., Metcalf, R. L., Carter, L., Brady, G., Blackhall, F. H., & Dive, C. (2014). Molecular analysis of circulating tumour cellsbiology and biomarkers. Nature Reviews. Clinical Oncology, 11(3), 129–144. doi:10.1038/nrclinonc.2013.253. Lianidou, E. S., Mavroudis, D., & Georgoulias, V. (2013). Clinical challenges in the molecular characterization of circulating tumour cells in breast cancer. British Journal of Cancer, 108(12), 2426– 2432. doi:10.1038/bjc.2013.265. Klein, C. A. (2009). Parallel progression of primary tumours and metastases. Nature Reviews Cancer, 9(4), 302–312. doi:10.1038/ nrc2627. Grade, M., Becker, H., Liersch, T., Ried, T., & Ghadimi, B. M. (2006). Molecular cytogenetics: genomic imbalances in colorectal cancer and their clinical impact. Cellular Oncology The Official Journal of the International Society for Cellular Oncology, 28(3), 71–84. Bruin, S. C., Klijn, C., Liefers, G. J., Braaf, L. M., Joosse, S. A., van Beers, E. H., et al. (2010). Specific genomic aberrations in primary colorectal cancer are associated with liver metastases. BMC Cancer, 10, 662. doi:10.1186/1471-2407-10-662. Pantel, K., Deneve, E., Nocca, D., Coffy, A., Vendrell, J. P., Maudelonde, T., et al. (2012). Circulating epithelial cells in patients

Cancer Metastasis Rev

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

with benign colon diseases. Clinical Chemistry, 58(5), 936–940. doi:10.1373/clinchem.2011.175570. Pantel, K., & Brakenhoff, R. H. (2004). Dissecting the metastatic cascade. Nature Reviews Cancer, 4(6), 448–456. doi:10.1038/ nrc1370. Weigelt, B., Glas, A. M., Wessels, L. F., Witteveen, A. T., Peterse, J. L., & Veer, L. J. (2003). Gene expression profiles of primary breast tumors maintained in distant metastases. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15901–15905. doi:10.1073/pnas.2634067100. Weigelt, B., Hu, Z., He, X., Livasy, C., Carey, L. A., Ewend, M. G., et al. (2005). Molecular portraits and 70-gene prognosis signature are preserved throughout the metastatic process of breast cancer. Cancer Research, 65(20), 9155–9158. doi:10.1158/0008-5472. CAN-05-2553. Shah, S. P., Morin, R. D., Khattra, J., Prentice, L., Pugh, T., Burleigh, A., et al. (2009). Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature, 461(7265), 809–813. doi:10.1038/nature08489. Schmidt-Kittler, O., Ragg, T., Daskalakis, A., Granzow, M., Ahr, A., Blankenstein, T. J., et al. (2003). From latent disseminated cells to overt metastasis: genetic analysis of systemic breast cancer progression. Proceedings of the National Academy of Sciences of the United States of America, 100(13), 7737–7742. doi:10.1073/pnas. 1331931100. Weckermann, D., Polzer, B., Ragg, T., Blana, A., Schlimok, G., Arnholdt, H., et al. (2009). Perioperative activation of disseminated tumor cells in bone marrow of patients with prostate cancer. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology, 27(10), 1549–1556. doi:10.1200/JCO.2008.17.0563. Husemann, Y., Geigl, J. B., Schubert, F., Musiani, P., Meyer, M., Burghart, E., et al. (2008). Systemic spread is an early step in breast cancer. Cancer Cell, 13(1), 58–68. doi:10.1016/j.ccr.2007.12.003. Rhim, A. D., Mirek, E. T., Aiello, N. M., Maitra, A., Bailey, J. M., McAllister, F., et al. (2012). EMT and dissemination precede pancreatic tumor formation. Cell, 148(1–2), 349–361. doi:10.1016/j. cell.2011.11.025. Kang, Y., & Pantel, K. (2013). Tumor cell dissemination: emerging biological insights from animal models and cancer patients. Cancer Cell, 23(5), 573–581. doi:10.1016/j.ccr.2013.04.017. Stoecklein, N. H., & Klein, C. A. (2010). Genetic disparity between primary tumours, disseminated tumour cells, and manifest metastasis. International Journal of Cancer/Journal International du cancer, 126(3), 589–598. doi:10.1002/ijc.24916. Horlings, H. M., Lai, C., Nuyten, D. S., Halfwerk, H., Kristel, P., van Beers, E., et al. (2010). Integration of DNA copy number alterations and prognostic gene expression signatures in breast cancer patients. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 16(2), 651–663. doi: 10.1158/1078-0432.CCR-09-0709. Joosse, S. A. (2012). BRCA1 and BRCA2: a common pathway of genome protection but different breast cancer subtypes. Nature Reviews Cancer, 12(5), 372. doi:10.1038/nrc3181-c2. author reply. Joosse, S. A., Brandwijk, K. I., Mulder, L., Wesseling, J., Hannemann, J., & Nederlof, P. M. (2011). Genomic signature of BRCA1 deficiency in sporadic basal-like breast tumors. Genes, Chromosomes & Cancer, 50(2), 71–81. doi:10.1002/gcc.20833. Lim, E., Vaillant, F., Wu, D., Forrest, N. C., Pal, B., Hart, A. H., et al. (2009). Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nature Medicine, 15(8), 907–913. doi:10.1038/nm.2000. Molyneux, G., Geyer, F. C., Magnay, F. A., McCarthy, A., Kendrick, H., Natrajan, R., et al. (2010). BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell Stem Cell, 7(3), 403–417. doi:10.1016/j.stem. 2010.07.010.

37.

38.

39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49.

50. 51. 52.

Zahl, P. H., Maehlen, J., & Welch, H. G. (2008). The natural history of invasive breast cancers detected by screening mammography. Archives of Internal Medicine, 168(21), 2311–2316. doi:10.1001/ archinte.168.21.2311. Stella, G. M., Senetta, R., Cassenti, A., Ronco, M., & Cassoni, P. (2012). Cancers of unknown primary origin: current perspectives and future therapeutic strategies. Journal of Translational Medicine, 10, 12. doi:10.1186/1479-5876-10-12. Suzuki, M., Mose, E. S., Montel, V., & Tarin, D. (2006). Dormant cancer cells retrieved from metastasis-free organs regain tumorigenic and metastatic potency. The American Journal of Pathology, 169(2), 673–681. doi:10.2353/ajpath.2006.060053. Tarin, D. (2012). Clinical and biological implications of the tumor microenvironment. Cancer Microenvironment, 5(2),95–112. doi: 10.1007/s12307-012-0099-6. Pantel, K., Schlimok, G., Braun, S., Kutter, D., Lindemann, F., Schaller, G., et al. (1993). Differential expression of proliferationassociated molecules in individual micrometastatic carcinoma cells. Journal of the National Cancer Institute, 85(17), 1419–1424. Janni, W., Vogl, F. D., Wiedswang, G., Synnestvedt, M., Fehm, T., Juckstock, J., et al. (2011). Persistence of disseminated tumor cells in the bone marrow of breast cancer patients predicts increased risk for relapse—a European pooled analysis. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 17(9), 2967–2976. doi:10.1158/1078-0432.CCR-10-2515. Paget, S. (1989). The distribution of secondary growths in cancer of the breast, 1889. Cancer Metastasis Reviews, 8(2), 98–101. Yumoto, K., Eber, M. R., Berry, J. E., Taichman, R. S., & Shiozawa, Y. (2014). Molecular pathways: niches in metastatic dormancy. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 20(13), 3384–3389. doi:10. 1158/1078-0432.CCR-13-0897. Braun, S., Kentenich, C., Janni, W., Hepp, F., de Waal, J., Willgeroth, F., et al. (2000). Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology, 18(1), 80–86. Braun, S., Vogl, F. D., Naume, B., Janni, W., Osborne, M. P., Coombes, R. C., et al. (2005). A pooled analysis of bone marrow micrometastasis in breast cancer. The New England Journal of Medicine, 353(8), 793–802. doi:10.1056/NEJMoa050434. Sanger, N., Effenberger, K. E., Riethdorf, S., Van Haasteren, V., Gauwerky, J., Wiegratz, I., et al. (2011). Disseminated tumor cells in the bone marrow of patients with ductal carcinoma in situ. International Journal of Cancer/Journal International du cancer, 129(10), 2522–2526. doi:10.1002/ijc.25895. Solakoglu, O., Maierhofer, C., Lahr, G., Breit, E., Scheunemann, P., Heumos, I., et al. (2002). Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. Proceedings of the National Academy of Sciences of the United States of America, 99(4), 2246–2251. doi:10.1073/pnas. 042372199. Meng, S., Tripathy, D., Frenkel, E. P., Shete, S., Naftalis, E. Z., Huth, J. F., et al. (2004). Circulating tumor cells in patients with breast cancer dormancy. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 10(24), 8152–8162. doi:10.1158/1078-0432.CCR-04-1110. Tabassum, D. P., & Polyak, K. (2015). Tumorigenesis: it takes a village. Nature Reviews Cancer, 15(8), 473–483. doi:10.1038/nrc3971. Navin, N. E., & Hicks, J. (2010). Tracing the tumor lineage. Molecular Oncology, 4(3), 267–283. doi:10.1016/j.molonc.2010.04.010. Navin, N., Kendall, J., Troge, J., Andrews, P., Rodgers, L., McIndoo, J., et al. (2011). Tumour evolution inferred by singlecell sequencing. Nature, 472(7341), 90–94. doi:10.1038/ nature09807.

Cancer Metastasis Rev 53.

De Roock, W., Claes, B., Bernasconi, D., De Schutter, J., Biesmans, B., Fountzilas, G., et al. (2010). Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. The Lancet Oncology, 11(8), 753–762. doi:10.1016/S1470-2045(10)70130-3. 54. Hannemann, J., Meyer-Staeckling, S., Kemming, D., Alpers, I., Joosse, S. A., Pospisil, H., et al. (2011). Quantitative highresolution genomic analysis of single cancer cells. PLoS ONE, 6(11), e26362. doi:10.1371/journal.pone.0026362. 55. Gasch, C., Bauernhofer, T., Pichler, M., Langer-Freitag, S., Reeh, M., Seifert, A. M., et al. (2013). Heterogeneity of epidermal growth factor receptor status and mutations of KRAS/PIK3CA in circulating tumor cells of patients with colorectal cancer. Clinical Chemsistry, 59(1), 252–260. doi:10.1373/clinchem.2012.188557. 56. Maley, C. C., Galipeau, P. C., Finley, J. C., Wongsurawat, V. J., Li, X., Sanchez, C. A., et al. (2006). Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nature Genetics, 38(4), 468–473. doi:10.1038/ng1768. 57. Notta, F., Mullighan, C. G., Wang, J. C., Poeppl, A., Doulatov, S., Phillips, L. A., et al. (2011). Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating cells. Nature, 469(7330), 362–367. doi:10.1038/nature09733. 58. Almendro, V., Cheng, Y. K., Randles, A., Itzkovitz, S., Marusyk, A., Ametller, E., et al. (2014). Inference of tumor evolution during chemotherapy by computational modeling and in situ analysis of genetic and phenotypic cellular diversity. Cell Reports, 6(3), 514– 527. doi:10.1016/j.celrep.2013.12.041. 59. Marusyk, A., Tabassum, D. P., Altrock, P. M., Almendro, V., Michor, F., & Polyak, K. (2014). Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature, 514(7520), 54–58. doi:10.1038/nature13556. 60. Calbo, J., van Montfort, E., Proost, N., van Drunen, E., Beverloo, H. B., Meuwissen, R., et al. (2011). A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell, 19(2), 244–256. doi:10.1016/j.ccr.2010.12.021. 61. Cayrefourcq, L., Mazard, T., Joosse, S., Solassol, J., Ramos, J., Assenat, E., et al. (2015). Establishment and characterization of a cell line from human circulating colon cancer cells. Cancer Research, 75(5), 892–901. doi:10.1158/0008-5472.CAN-14-2613. 62. Aceto, N., Bardia, A., Miyamoto, D. T., Donaldson, M. C., Wittner, B. S., Spencer, J. A., et al. (2014). Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell, 158(5), 1110–1122. doi:10.1016/j.cell.2014.07.013.

63.

Kreso, A., O’Brien, C. A., van Galen, P., Gan, O. I., Notta, F., Brown, A. M., et al. (2013). Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer. Science, 339(6119), 543–548. doi:10.1126/science.1227670. 64. Pao, W., Miller, V. A., Politi, K. A., Riely, G. J., Somwar, R., Zakowski, M. F., et al. (2005). Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Medicine, 2(3), e73. doi:10.1371/journal.pmed.0020073. 65. Heitzer, E., Auer, M., Gasch, C., Pichler, M., Ulz, P., Hoffmann, E. M., et al. (2013). Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. Cancer Research, 73(10), 2965–2975. doi:10.1158/00085472.CAN-12-4140. 66. Wu, C. C., Maher, M. M., & Shepard, J. A. (2011). Complications of CT-guided percutaneous needle biopsy of the chest: prevention and management. AJR. American Journal of Roentgenology, 196(6), W678–W682. doi:10.2214/AJR.10.4659. 67. Bednarz-Knoll, N., Alix-Panabieres, C., & Pantel, K. (2012). Plasticity of disseminating cancer cells in patients with epithelial malignancies. Cancer Metastasis Reviews, 31(3–4), 673–687. doi: 10.1007/s10555-012-9370-z. 68. Yu, M., Bardia, A., Wittner, B. S., Stott, S. L., Smas, M. E., Ting, D. T., et al. (2013). Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science, 339(6119), 580–584. doi:10.1126/science.1228522. 69. Guzvic, M., Braun, B., Ganzer, R., Burger, M., Nerlich, M., Winkler, S., et al. (2014). Combined genome and transcriptome analysis of single disseminated cancer cells from bone marrow of prostate cancer patients reveals unexpected transcriptomes. Cancer Research, 74(24), 7383–7394. doi:10.1158/0008-5472.CAN-140934. 70. Miyamoto, D. T., Zheng, Y., Wittner, B. S., Lee, R. J., Zhu, H., Broderick, K. T., et al. (2015). RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science, 349(6254), 1351–1356. doi:10.1126/science.aab0917. 71. Miyamoto, D. T., Lee, R. J., Stott, S. L., Ting, D. T., Wittner, B. S., Ulman, M., et al. (2012). Androgen receptor signaling in circulating tumor cells as a marker of hormonally responsive prostate cancer. Cancer Discovery, 2(11), 995–1003. doi:10.1158/2159-8290.CD12-0222. 72. Almendro, V., Kim, H. J., Cheng, Y. K., Gonen, M., Itzkovitz, S., Argani, P., et al. (2014). Genetic and phenotypic diversity in breast tumor metastases. Cancer Research, 74(5), 1338–1348. doi:10. 1158/0008-5472.CAN-13-2357-T.