Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression.

Analyses of the cellular and viral Kirsten ras genes (c-Ki-ras and v-Ki-ras, respectively) during malignant tumor progression were performed by using Kirsten murine sarcoma virus-transformed BALB/c 3T3 cells that harbor a replication-defective provirus. After injection into athymic nude mice by four different routes, primary tumors and secondary lung metastases were isolated, adapted to in vitro growth, and analyzed for DNA levels and mRNA expression of both genes for comparison with the originally injected transformed cells and untransformed 3T3 cells. For all tumors (primary or secondary), the v-Ki-ras gene was amplified and v-Ki-ras mRNA expression was highly elevated above that observed in the original transformed cell population. In two of five lung metastases from the i.v. and footpad injection routes, rearranged Ki-ras DNA sequences were observed. Micrometastases from the s.c. route of injection did not display these alterations. Injection of footpad lung tumor cells with rearrangements into a second group of animals led to multiple lung metastases with even further rearrangements correlating with more effective lung colonization/growth ability (overt lung tumors in five of eight animals less than 20 days after injection). However, reinjection of an i.v. lung tumor with rearranged Ki-ras led to no further rearrangements in the lung microfoci tumors isolated greater than 40 days after injection. These data suggest (i) the significance of amplification and elevated expression of v-Ki-ras in tumor formation, (ii) correlation of this amplification with more effective tumor progression, and (iii) the selective advantage that cells with Ki-ras DNA sequence additions have in the formation of overt lung tumors.     

Is tail vein injection a relevant breast cancer lung metastasis model?

Background

Two most commonly used animal models for studying breast cancer lung metastasis are: lung metastasis after orthotopic implantation of cells into the mammary gland, and lung implantations produced after tail vein (TV) injection of cells. Tail vein injection can produce lung lesions faster, but little has been studied regarding the differences between these tumors, thus, we examined their morphology and gene expression profiles.

Methods

Syngeneic murine mammary adenocarcinoma, 4T1-luc2 cells, were implanted either subcutaneously (Sq), orthotopically (OS), or injected via TV in Balb/c mice. Genome-wide microarray analyses of cultured 4T1 cells, Sq tumor, OS tumor, lung metastases after OS (LMet), and lung tumors after TV (TVt) were performed 10 days after implantation.

Results

Bioluminescence analysis demonstrated different morphology of metastases between LMet and TVt, confirmed by histology. Gene expression profile of cells were significantly different from tumors, OS, Sq, TVt or LMet (10,350 probe sets; FDR≤1%; P<0.0001). Sq tumors were significantly different than OS tumors (700 probe sets; FDR≤15%; P<0.01), and both tumor types (Sq and OS) were significantly different than LMet (1,247 probe sets; >1.5-fold-change; P<0.01), with no significant difference between TVt and LMet.

Conclusions

There were significant differences between the gene profiles of cells in culture and OS versus LMet, but there were no differences between LMet versus TVt. Therefore, the lung tumor generated by TVt can be considered genetically similar to those produced after OS, and thus TVt is a relevant model for breast cancer lung metastasis.KEY WORDS : Breast cancer, lung metastasis, animal model, microarray, metastasis model     

Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis

Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.Lung cancer remains the major cause of death from cancer worldwide. The two types of lung cancer are non-small-cell lung cancers (NSCLCs; 80% of all lung cancers), which include adenocarcinomas, squamous cell carcinomas, and large cell carcinomas, and small-cell lung cancers (20%, exhibiting neuroendocrine features). The average 5-y survival rate for NSCLC is only 16% (1). Most NSCLC patients have advanced disease at diagnosis; 22% have regional lymph node metastases, and 56% have distant metastases in the brain, bone, liver, or adrenal glands (1). Surgery or therapies that treat primary lung tumors rarely prevent metastases (1). Understanding the mechanisms of NSCLC metastasis is crucial for the development of new therapies to improve survival for lung cancer patients.Tropomyosin-related kinase B (TrkB; also called NTRK2), a neurotrophin receptor, is important for neural development and is an independent prognostic marker in many tumor types (2). TrkB-expressing neural precursors migrate from their proliferative zone toward a gradient of the TrkB ligand, brain-derived neurotrophic factor (BDNF), which is made near the site of residence of the mature neurons (3). High levels of TrkB are correlated with poor patient prognosis in neuroblastomas and ovarian, pancreatic, colon, prostate, and gastric cancers (2). TrkB was overexpressed in ovarian adenocarcinoma metastases compared with primary lesions (4). TrkB is also a supressor of anoikis in cell lines (5). Finally, overexpression of TrkB and BDNF in nonmalignant rat intestinal epithelial cells promoted metastasis from s.c. injection sites to the lungs (6).Despite these clues that TrkB regulates parts of the metastatic cascade in other tumor types, the role of TrkB in NSCLC metastasis remains unclear. TrkB immunoreactivity was correlated with advanced stage disease in lung squamous cell carcinomas, yet was also correlated with better survival (7). In contrast, TrkB expression was associated with worse survival in a panel of NSCLC samples containing squamous cell carcinomas, adenocarcinomas, and large-cell neuroendocrine carcinomas (8). Despite the evidence that TrkB is important for metastasis of cell lines, it has not been established that TrkB is required for NSCLC metastatic activity in endogenous tumors, and only xenograft models have been used to investigate TrkB function in other tumor types.We developed an in vitro system to identify factors crucial for lung cancer metastasis. In this study, we determined that BDNF secreted by lymph node fibroblasts induces migration of lung cancer cells. TrkB was required for migration of lung adenocarcinoma cancer cell lines and their metastatic capacity in vivo. Loss of TrkB significantly reduced lymph node metastases in a lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss. TrkB expression was regulated by hypoxia-inducible factor-1 (HIF-1), and TrkB was required for Akt activation during lung tumor cell migration. TrkB expression was correlated with a higher stage and with observed metastasis in lung cancer patient samples. Our work demonstrates that TrkB is crucial for the migratory steps in lung adenocarcinoma metastasis.     

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed “tumor self-seeding.” Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as “tumor self-targeting.” For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell–mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.A hallmark of cancer is the dissemination of tumor cells into the vascular system and colonization of distant organs to form metastatic foci. Recent studies showed that circulating cancer cells can traffic from primary or metastatic lesions to the peripheral blood and then back to their tumor of origin through a process termed “self-seeding” (1, 2). This process is proposed to mediate cancer progression by recruiting circulating tumor cells capable of surviving under harsh conditions in the bloodstream, thereby promoting both primary tumor growth and secondary metastatic dissemination (1, 3). Such homing properties may be facilitated by the presence of blood vessels with altered vascular endothelial barrier function in tumors (2, 4) and by the presence of a favorable tumor microenvironment (1).This unique concept provides a rationale for exploiting cancer cells (5) as vehicles to deliver therapeutic agents to tumors, referred to hereafter as (“tumor self-targeting”). Indeed, oncolytic viruses incorporated within cancer cells have been shown to target lung metastases, being protected from circulating antibodies (5–7). Thus, we reasoned that “armed” tumor cells genetically manipulated to express anticancer cytokines and subsequently administered into the bloodstream should be able to deliver such factors focally to both primary and metastatic lesions as a therapeutic strategy. This delivery method may circumvent toxicity and activation of systemic counterregulatory mechanisms, which currently cause major limitations for cytokine biotherapy against cancer (8). To address this hypothesis experimentally, we engineered murine mammary adenocarcinoma, melanoma, and lung carcinoma cells to produce and release tumor necrosis factor alpha (TNF), a cytokine that damages the tumor vascular endothelia and has anticancer activity (9–11). The rationale for choosing this cytokine is based on our previous work that demonstrated ligand-based delivery of extremely low concentrations of TNF to tumor blood vessels markedly inhibits tumor growth in various xenograft (12–14) experimental mouse tumor models, and in a variety of native tumors in pet dogs (15). We show that systemic administration of several types of TNF-expressing cancer cells in syngeneic and nonsyngeneic tumor-bearing mice inhibits the growth of primary and metastatic cancers. We provide evidence that this effect is due to specific targeting to tumors, whereby injected cells accumulate and locally release TNF, resulting in tumor vasculature damage and tumor cell apoptosis.     

Predilection of contralateral upper lung metastasis in upper lobe lung adenocarcinoma patients

Background

Lung cancer with lung to lung metastasis is common. The objective of this study was to investigate the association among the distribution of contralateral lung metastases versus primary lung tumor location, clinical characteristics, and epidermal growth factor receptor (EGFR) mutations status.

Methods

The study included treatment-naïve stage IV lung adenocarcinoma with contralateral lung metastases from 2012 through 2013.

Results

In total, 103 patients were enrolled after excluding lung cancer with histology other than adenocarcinoma, synchronous multiple primary lung cancers, or other active malignancy. The median age was 65 years (range, 28–93 years); 47 male patients (45.6%); 69 non-smoker (NS) patients (67.0%); 68 Eastern Cooperative Oncology Group performance status (ECOG PS) 0–1 patients (66.0%); 38 M1a patients (38.9%); and 60 EGFR mutation patients (58.3%). There were 51 cases (49.5%) with primary lung cancer located over upper lobes. Among them, 36 (70.6%) had contralateral upper lung predominance metastasis, 9 (17.6%) had lower lung predominance, and 6 (11.8%) had equal distribution. Among the 52 lower lobe tumors, 17 (32.7%), 19 (36.5%), and 16 (30.8%) had upper, lower lung predominance, and equal distribution metastasis, respectively. Univariate analysis showed only male gender and primary tumor location over upper lobes were significantly associated with contralateral upper lung predominance metastases. After multivariate analysis, only primary tumor location over upper lobes was significantly associated with contralateral upper lung predominance metastases (adjusted OR 5.49, 95% CI, 2.15–14.03, P<0.001).

Conclusions

Upper lobe lung adenocarcinoma was significantly associated with contralateral upper lung predominance metastases. Further research is needed to elucidate the mechanisms underlying this phenomenon.     

Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.     

Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination

Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.As in many other solid tumors, melanoma metastases often first present in lymph nodes in the draining area of the primary, whereas distant metastases tend to appear later (1). The conclusion that melanoma follows a linear progression from primary tumor to regional to distant metastases has supported preemptive surgical removal of regional lymph nodes with curative intent (2). However, several observations suggest that distant metastases are seeded early, contemporaneously with regional metastases. Patients who undergo resection of lymph node basins harboring metastasis do not experience a significantly extended life expectancy (3, 4). Furthermore, circulating melanoma cells were detected in the blood of 26% of patients who only have metastases detected regionally (5, 6).Melanoma, like other cancers, arises and evolves through the accumulation of genetic alterations within tumor cells (7–9). Comparing somatic mutations in primary tumor and regional and distant metastases from the same patient can provide insight into the phylogenetic relationships between these distinct tumor cell populations and the order of metastatic dissemination (8, 10). These analyses may also establish whether cells in the primary tumor that metastasize acquired this ability to disseminate and seed other anatomic sites by a newly acquired genetic alteration, or whether metastatic colonization is simply a stochastic process of which all cells in the primary are capable but few succeed.Using whole-exome sequencing (for discovery) and targeted sequencing (for validation), we analyzed mutation patterns of primary melanomas and two or more metastases in each of eight patients (Datasets S1–S8) to determine their phylogenetic relationships.     

Gene Therapy of Patient-Derived T Lymphocytes to Target and Eradicate Colorectal Hepatic Metastases

PURPOSE: The overall aim of this study was to develop a novel treatment for colorectal cancer based on the use of gene therapy. Genetic modification of T lymphocytes has been used to specifically target and kill tumor cell lines directly. To test the efficacy of this method with clinically relevant materials, this study investigated the potential of T lymphocytes derived from patients with advanced colorectal disease to target autologous primary tumor material. METHODS: T lymphocytes isolated preoperatively were modified genetically with recombinant retroviruses encoding CD3-based chimeric immune receptors and were tested for functional activity against freshly isolated autologous tumor cells harvested from hepatic colorectal metastases. RESULTS: Patient-derived T cells were successfully transduced, and chimeric immune receptor expression was confirmed. Carcinoembryonic antigen expression on freshly isolated colorectal tumor cells was also demonstrated by molecular and immunohistochemical techniques. T cells expressing the anticarcinoembryonic antigen receptor were specifically activated by coculture with disaggregated or intact, diced tumor, whereas control non-carcinoembryonic antigen-targeted T-cell populations failed to activate. CONCLUSIONS: These results indicate that gene-targeted primary T lymphocytes depict specific functional activity against autologous colorectal tumor cells. This evidence indicates that chimeric immune receptor-expressing T cells may be able to circumvent the mechanisms used by tumor cells to avoid immune cell activity in vivo. This study emphasizes the potential of this approach as a therapy for carcinoembryonic antigen-expressing primary colorectal tumor and its metastases.     

From the Cover: Nontoxic radioactive Listeriaat is a highly effective therapy against metastatic pancreatic cancer

No significant improvement in therapy of pancreatic cancer has been reported over the last 25 y, underscoring the urgent need for new alternative therapies. Here, we coupled a radioisotope, ¹⁸⁸Rhenium, to an attenuated (at) live Listeria monocytogenes (Listeria^at) using Listeria-binding antibodies, thus creating a unique radioactive Listeria^at (RL). We then demonstrated in a highly metastatic pancreatic mouse tumor model (Panc-02) that RL delivered radioactivity to the metastases and less abundantly to primary tumors in vivo, without harming normal cells. This result was possible because Listeria^at was efficiently cleared by the immune system in normal tissues but not in the heavily immune-suppressed microenvironment of metastases and primary tumor. Multiple treatments with low doses of the RL resulted in a dramatic decrease in the number of metastases (∼90%) compared with control groups in the Panc-02 model. This is the first report of using live attenuated bacteria delivering a highly radioactive payload to the metastases, resulting in killing tumor cells in vivo without harming normal cells. The nontoxic RL treatment is attractive for clinical development as a therapy to prevent pancreatic cancer recurrence and metastases.Pancreatic ductal adenocarcinoma, synonymous to pancreatic cancer, is the fourth leading cause of cancer deaths. The so-called silent killer is characterized by its metastatic behavior before the primary tumor can be detected, resulting in a 5-y survival rate of only 4%. Current cancer treatments—i.e., surgery followed by radiation and/or chemotherapy—are ineffective, particularly against liver metastases. Gemcitabine and erlotinib, Food and Drug Administration-approved drugs for pancreatic cancer treatment, improve median survival by ∼6 mo in patients with advanced-stage disease (1–3), emphasizing the need for new alternative therapies for metastatic pancreatic cancer.For any anticancer approach to be effective, it needs to target metastases and/or remaining tumor cells after primary therapeutic intervention. Indeed, in most cases, cancer therapy is now highly effective in eradicating primary tumors through combinations of surgery, radiation, and adjuvant therapy. The reason that cancer remains such a formidable health problem is its capacity to recur in the form of widespread metastases, often with a fatal consequence. In a previous study, we found that a highly attenuated bacterium, Listeria monocytogenes (Listeria^at), which was originally used to deliver tumor-associated antigens into antigen-presenting cells, also infected tumor cells in vitro and in vivo (4). Although Listeria^at was efficiently cleared by the immune system in the normal tissues within 3–5 d, immune suppression in the tumor microenvironment allowed these bacteria to accumulate in metastases and primary tumors and to kill tumor cells through high levels of reactive oxygen species (ROS) (4). Based on these results we hypothesized that Listeria^at could be used to deliver anticancer agents, such as therapeutic radionuclides (emitting cytocidal radiation such as beta-particles), specifically to the microenvironment of metastases and primary tumors and into tumor cells.Targeted radionuclide therapy has proven successful in the treatment of several types of cancer and currently employs radiolabeled small molecules, monoclonal antibodies (Abs), peptides, and other tumor-targeting vehicles (5). The radioactive particles emitted by the radionuclides physically destroy the cancerous cells, and such therapies are not subject to multidrug-resistance mechanisms. There have been attempts to use targeted radionuclide therapy in form of radiolabeled tumor-specific Abs (radioimmunotherapy) for treatment of pancreatic cancer. However, radioimmunotherapy of pancreatic cancer has shown very modest results both preclinically (6–8) and in cancer patients with unresectable liver metastases (9). Obviously, new choices of targeting vehicles are needed to make targeted radionuclide therapy successful in the treatment of pancreatic cancer. In this regard, Listeria^at provides an attractive system for the delivery of radionuclides into the microenvironment of metastases and primary tumors. Here we show that live attenuated Listeria^at coupled with radionuclide ¹⁸⁸Rhenium (¹⁸⁸Re) is highly effective, particularly against metastases, in a mouse model of pancreatic cancer (Panc-02 model) without appreciable side effects.     

Anticoagulants and experimental metastases-evaluation of antimetastatic effects in different model systems

Summary Intravenous tumor cell injection and spontaneously metastasising transplantable tumors have been used as models for haematogenous tumor spread. To evaluate the validity of these two experimental approaches for the study of blood coagulability and metastases, the effect of anticoagulants on secondary tumor growth was investigated in both models and the results were compared. The B16 melanoma and the Lewis lung carcinoma in C57BL mice were employed throughout the study, and anticoagulation was rendered through the use of phenprocoumon, heparin and ancrod. All anticoagulants were capable of reducing lung colonies after i.v. tumor cell injection, whereas only phenprocoumon significantly diminished the formation of spontaneous metastases. A review of the current literature on anticoagulants and tumor dissemination and the observations described lead to the following conclusions: (1) Results from studies with i.v. introduced tumor cells cannot be extrapolated to spontaneously metastasising tumors. (2) Spontaneously metastasising tumors represent the preferable model for the study of antimetastatic effects of anticoagulants. (3) Little evidence exists to support the concept of the pathogenetic role of fibrin formation in the establishment of spontaneous metastases from blood-borne tumor cells. (4) Coumarin derivatives are potent antimetastatic drugs, their mode of action appears to be independent of their anticoagulant activity.This study was performed in a programme of the EORTC Metastasis Project Group. Parts of the experiments were carried out at the West German Cancer Centre, University of Essen, FRG. Dr. P. Hilgard was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, FRG (Hi 213/3-4)     

Analysis of metastatic spread and growth of tumor cells in mice with depressed natural killer activity by anti-asialo GMl antibody or anticancer agents

Summary The mechanism of artificial and spontaneous metastases of tumor was analyzed in B16 melanoma cells and C57BL/6 mice by using anti-asialo GM1 antibody and anticancer agents. Single administrations of 500 g anti-asialo GM1 antibody resulted in significantly decreased NK activity in spleen cells of C57BL/6 mice, lasting 10 days from the day following administration. Treatment with anti-asialo GM1 antibody never decreased the function of T lymphocytes measured by blastogenesis with phytohemagglutinin or T cell growth factor. The tumoricidal functions of activated macrophages but not of resident macrophages were decreased by in vivo treatment with anti-asialo GM1 antibody.The anti-asialo GM1 antibody was evaluated in terms of the enhancing effect on pulmonary metastases with regard to the timing of administration. Treatment with anti-asialo GM1 antibody 1 day before or on the day of tumor inoculation resulted in a substantial increase in the number of artificial pulmonary metastases. In the experimental system of spontaneous metastases, anti-asialo GM1 antibody most effectively increased the number of pulmonary metastases when administered 1–2 weeks before the removal of primary tumor, when the tumor cells are thought to be released into blood circulation from the primary site. In addition, accelerated growth of transplanted tumors at the primary site was observed in mice treated with anti-asialo GM1 antibody. These results strongly suggest that anti-asialo GM1 antibody enhances the incidence of in vivo tumor metastases and the growth of transplanted tumor mainly by suppressing the function of NK cells.The maximum effective dose (MED) of mitomycin C or its derivative (M-83) suppressed NK activity significantly, and pretreatment with these anticancer agents enhanced the growth of the artificial pulmonary and liver metastases. In contrast, the MED of cDDP showed no effect on the NK activity or the numbers of pulmonary and liver metastases. These results indicate that the depression of NK activity induced by chemotherapy results in the promotion of metastatic disease.From these studies it can be concluded that NK cells have a key role in the control of metastases of malignant disease, and that support of NK activity is very important for the prevention of metastases.     

Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling

We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.Large-scale sequencing analyses of solid cancers have identified extensive tumor heterogeneity within individual primary cancers (1). Recent studies indicate that such tumoral heterogeneity is associated with heterogeneous protein function, which fosters tumor adaptation, treatment resistance, and failure through Darwinian selection (2–4). Cancer stem cells are a subpopulation of cells within the primary tumor responsible for tumor initiation and metastases (5–9). Three groups have recently independently provided functional evidence for the presence of cancer stem cells by lineage-tracing experiments (10–12). These observations suggest that these subpopulations of cancer stem cells (CSCs) within the bulk primary tumor are resistant to conventional therapies through different adaptive mechanisms with the potential for self-renewal and metastases (7, 13, 14). However, few studies have determined the genetic profile of the cells that escape the primary cancer and evolve in distant metastatic sites (1). Additionally, no large-scale sequencing studies of metastases have been conducted because the majority of patients are treated with systemic therapies and not surgery.Tumor clonal heterogeneity within a primary tumor may in part be explained by hypoxic regions within the bulk tumor that have been correlated with invasiveness, therapeutic resistance, and metastasis (15–18). Cancer stem cells have been found to reside near hypoxic regions in some solid cancers (19–21). We have previously published a 477-gene tumorigenic signature by isolating breast cancer stem cells (BCSCs) derived from patient biopsies (22). Here, we have identified two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2), by selective shRNA knockdown of genes from this tumorigenic signature, that impact breast cancer stem cell self-renewal and lung metastases. Analysis of 53 patient lung metastases confirmed damaging mutations in RPL39 and MLF2 in a significant number of samples, which conferred a gain-of-function phenotype. These mutations were statistically associated with shorter median time to distant relapse. We further describe a common mechanism of action through nitric oxide synthase signaling that is regulated by hypoxia.     

Efficacy of lymph node dissection for each station based on esophageal tumor location

Background

The area of nodal dissection should be modified by the location of the primary tumor in an individual patient. The purpose of this study was to evaluate the efficacy of lymph node dissection based on station by the location of the primary tumor based on a multi-institutional nationwide registry of esophageal cancer.

Methods

The study group comprised 1295 patients who underwent R0 resection and three-field esophagectomy. The Efficacy Index (EI) was calculated by multiplying the incidence of metastases to a station and the 5-year survival rate of patients with metastases to that station, by tumor location.

Results

There were 550 patients without nodal metastases and 745 patients with them. In patients with upper tumors, the EIs of recurrent nerve nodes, cervical paraesophageal nodes and supraclavicular nodes were highest. In patients with middle tumors, the EIs of recurrent nerve nodes, cardiac nodes and lesser curvature nodes were highest, and the EIs of supraclavicular nodes and cervical paraesophageal nodes were not negligible. In patients with lower tumors, the EIs of cardiac nodes, lesser curvature nodes and left gastric artery nodes were highest, and the EIs of recurrent nerve nodes were also high.

Conclusion

The EIs of certain node stations were different by location of the primary tumor. Node stations for dissection should be modified by the location of the primary tumor. For upper and middle esophageal tumors, the three-field approach is recommended. Dissection of the upper mediastinum is recommended for patients with lower esophageal tumors.

     

Combination OX40 agonism/CTLA-4 blockade with HER2 vaccination reverses T-cell anergy and promotes survival in tumor-bearing mice

Immunotherapy is gathering momentum as a primary therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only a subset of patients. Combination therapies targeting multiple pathways can augment an immune response to improve survival further. Here, we demonstrate that dual aOX40 (anti-CD134)/aCTLA-4 (anti–cytotoxic T-lymphocyte–associated protein 4) immunotherapy generated a potent antigen-specific CD8 T-cell response, enhancing expansion, effector function, and memory T-cell persistence. Importantly, OX40 and CTLA-4 expression on CD8 T cells was critical for promoting their maximal expansion following combination therapy. Animals treated with combination therapy and vaccination using anti–DEC-205 (dendritic and epithelial cells, 205 kDa)–HER2 (human epidermal growth factor receptor 2) had significantly improved survival in a mammary carcinoma model. Vaccination with combination therapy uniquely restricted Th2-cytokine production by CD4 cells, relative to combination therapy alone, and enhanced IFNγ production by CD8 and CD4 cells. We observed an increase in MIP-1α (macrophage inflammatory protein-1α)/CCL3 [chemokine (C-C motif) ligand 3], MIP-1β/CCL4, RANTES (regulated on activation, normal T-cell expressed and excreted)/CCL5, and GM-CSF production by CD8 and CD4 T cells following treatment. Furthermore, this therapy was associated with extensive tumor destruction and T-cell infiltration into the tumor. Notably, in a spontaneous model of prostate adenocarcinoma, vaccination with combination therapy reversed anergy and enhanced the expansion and function of CD8 T cells recognizing a tumor-associated antigen. Collectively, these data demonstrate that the addition of a vaccine with combined aOX40/aCTLA-4 immunotherapy augmented antitumor CD8 T-cell function while limiting Th2 polarization in CD4 cells and improved overall survival.Promoting a robust CD8 T-cell response is vital for the generation of an effective antitumor immune response. However, immunosuppressive mechanisms used by the tumor result in the exhaustion of tumor-infiltrating lymphocytes (TIL), leading to cytotoxic T-cell anergy and dysfunction. To overcome this dysfunction, investigators have had considerable success using immune checkpoint inhibitors, such as aCTLA-4 (cytotoxic T-lymphocyte–associated protein 4) mAbs, to promote T-cell function. CTLA-4, a negative regulatory molecule on the surface of T cells, is indispensable for preventing the expansion and activation of autoreactive T cells. Inhibition of this surface receptor using antagonist aCTLA-4 mAb–augmented effector CD4 and CD8 T-cell responses and inhibited or reduced the suppressive function of Treg cells (1–5). However, only a subset of patients treated with aCTLA-4 mAb exhibit an objective clinical response (6).Checkpoint blockade targets T-cell coinhibitory molecules, but other strategies targeting costimulatory molecules, such as the TNF receptor family member OX40 (CD134), have demonstrated success in promoting tumor regression in a wide variety of preclinical models. Treatment with an agonist aOX40 mAb directly stimulated CD4 and CD8 T cells and induced their expansion, differentiation, and up-regulation of prosurvival molecules (7–12). Moreover, OX40 ligation promoted the generation of long-lived memory CD8 T cells and enhanced their function. Recent data indicate that combined aOX40/aCTLA-4 therapy induced a robust effector CD4 and CD8 T-cell response necessary for tumor regression and significantly improved the survival of tumor-bearing hosts relative to therapy with either agent alone (13). Despite this success, the response to combination aOX40/aCTLA-4 treatment was greatly diminished in more established tumors. This therapy may be unable to overcome T-cell anergy in more established immunosuppressive tumor microenvironments, possibly because it is ineffective at specifically targeting and expanding tumor-reactive T cells and relies on limited or defective endogenous priming by dendritic cells (14). Currently, clinically tested vaccines targeting cross-presenting dendritic cells [i.e., anti–DEC-205 (dendritic and epithelial cells, 205 kDa) mAb conjugated to tumor antigens] have demonstrated promise by priming a more robust cytotoxic T-cell response (15–19). The possibility remains that increased Th2-cytokine production by CD4 T cells following combination therapy may reduce its therapeutic efficacy, because inhibition of IL-4 with aOX40/aCTLA-4 treatment significantly improved survival (13). Others have shown that a dominant Th2 cytokine response reduced the efficacy of treatment, whereas a Th1-skewed immune response resulted in more favorable outcomes (20–23).We hypothesized that dendritic cell-targeted vaccination against a tumor-associated antigen in conjunction with combination aOX40/aCTLA-4 mAb immunotherapy would be sufficient to promote a cytotoxic antitumor T-cell response, redirect a Th2 bias in CD4 T cells, and improve survival in mice with established tumors. Here, we report that vaccination using anti–DEC-205/HER2 (human epidermal growth factor receptor 2) mAb along with combination aOX40/aCTLA-4 mAbs significantly expanded effector CD8 T cells, resulting in a more favorable Th1 cytokine profile and inducing a critical accumulation of effector T cells in the tumor, with increased tumor-free survival relative to either therapy alone. Moreover, combination therapy with vaccination reversed T-cell anergy and promoted a robust effector T-cell response to a tumor-associated antigen in a spontaneous adenocarcinoma model.     

Renal Cell Carcinoma,Metastatic to the Left Ventricle

Left ventricular metastases from renal cell carcinoma without vena caval or right atrial involvement are extremely rare. Herein, we present the case of a 69-year-old man who had undergone radical nephrectomy for renal cell carcinoma in 1984. Eighteen years thereafter, we discovered metastatic disease in his left ventricle.When the metastasis was identified, the patient had no symptoms other than shortness of breath. He underwent surgical removal of a highly vascular mass from the left ventricular wall and resection of a nodule in the upper right pulmonary lobe. Upon pathologic examination, both tumors were metastatic renal cell carcinomas. The patient recovered uneventfully and was free of cardiac recurrence more than 6 years after the surgery. We describe our treatment of this patient and discuss some current approaches to the treatment of renal cell carcinoma that has metastasized to the heart.Key words: Carcinoma, renal cell/complications/pathology/secondary/surgery; coronary disease/etiology/surgery; heart neoplasms/diagnosis/secondary; heart ventricles; neoplasm invasiveness; time factors; treatment outcomeCardiac tumors are extremely rare, and metastatic disease that involves the heart is 20 to 40 times more frequent than are primary cardiac malignancies.¹ The most common secondary tumors of the heart originate from leukemia, melanoma, lung cancer, breast cancer, and lymphoma. Cardiac metastases from renal cell carcinoma (RCC), which are extremely rare, usually occur in either of 2 circumstances. First, advanced RCC characteristically extends into the renal vein and the inferior vena cava in 5% to 15% of patients, and into the right atrium in about 1% of patients, thereby obstructing venous return to the heart (1).^2,3 Second, there can be a primary tumor that metastasizes to the heart, which occurs in 10% to 20% of patients who are dying of widespread, systemic RCC. However, in the absence of either direct vena caval extension or systemic disease, involvement of the heart is extremely rare, with only 1 known report thereof in the medical literature.⁴ Here, we present the case of a 69-year-old man who experienced metastasis of RCC to the left ventricle and to the upper right lobe of the lung. We discuss our treatment of this patient, along with current surgical and therapeutic approaches to the treatment of RCC that has metastasized to the heart.     

Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells

Impressive responses have been observed in patients treated with checkpoint inhibitory anti–programmed cell death-1 (PD-1) or anti–cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti–PD-1 and anti–CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.The mammalian immune system is delicately regulated, allowing it to mount an effective attack against foreign invaders such as bacteria and viruses with minimal bystander casualties. This requires functionally redundant regulatory mechanisms to ensure safety (1–3). Cancers appear able to hijack these mechanisms to avoid immune destruction. Several of the regulatory mechanisms exploited by cancer have been identified. These include regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages and neutrophils, immune checkpoint pathways, and immunosuppressive cytokines (4–8). Most recently, the checkpoints guarded by the programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) receptors have been under intense investigation because of the availability of antibodies that can inhibit their function. Recent clinical trials with anti–CTLA-4, anti–PD-1, and anti–PD-L1 monoclonal antibodies showed remarkable therapeutic responses (9–12), underscoring the idea that disruption of immune checkpoints can be therapeutically useful. However, the objective responses were observed in a minority of the treated patients and tumor types, and the reasons why certain tumors respond and others do not are mysterious. CT26 and 4T1 are among the most popular syngeneic tumor models used for assessing novel therapeutic approaches. CT26 was derived from an undifferentiated colorectal carcinoma induced in a BALB/c mouse by repeated intrarectal instillations of N-nitroso-N-methylurethan and shown to be modestly immunogenic (13, 14), whereas 4T1 originated from a spontaneous mammary tumor in a BALB/c mouse (15). 4T1 is poorly immunogenic and highly metastatic, characteristics shared with advanced human cancers (16). Despite the extensive use of these tumor cell lines in cancer research, little genetic characterization is available for either of them.In the current study, we evaluated both models with respect to their responses to the immune checkpoint inhibitors alone and combined with other agents. We also determined the sequences of their coding genes and mutant peptides that bind to the class I major histocompatibility complex (MHC-I). We found that the more immunogenic CT26 cells had considerably more mutations than 4T1 cells, and that the majority of the tumors derived from CT26 cells could be cured by anti–PD-1 and anti–CTLA-4 antibodies. In contrast, 4T1-derived tumors could not generally be cured by these antibodies. Mechanistic studies led to the conclusion that MDSCs were interfering with our therapeutic attempts; reduction of these MDSCs allowed frequent cures of 4T1 tumors by PD-1/CTLA-4 inhibition, even when they were advanced and metastatic.     

Tumor Budding as an Indicator of Isolated Tumor Cells in Lymph Nodes From Patients With Node-Negative Colorectal Cancer

PURPOSE Isolated tumor cells are often found in the regional lymph nodes of colorectal cancer, although their prognostic significance has not been established yet. This study was performed to investigate the correlation between the presence of isolated tumor cells in lymph nodes and the histopathologic characteristics of colorectal cancers and, thus, to determine which factors are associated with isolated tumor cells.METHODS We used immunohistochemistry with anticytokeratin antibody to examine 2,784 lymph nodes in 109 patients with node-negative colorectal cancers. The clinicopathologic features of the tumors with isolated tumor cells were compared with those without isolated tumor cells. The frequency, number, and level of the isolated tumor cells also were assessed.RESULTS Isolated tumor cells were detected in 335 lymph nodes (12 percent) from 71 patients (65.1 percent). Those tumors having isolated tumor cells in lymph nodes, compared with those not having isolated tumor cells, were characterized by large tumor size, high T stage (pT3 and pT4), angiolymphatic invasion, perineural invasion, absence of peritumoral lymphocytic response, microsatellite instability-negative phenotype, and tumor budding. Multivariate analysis showed that those factors independently associated with the presence of isolated tumor cells were high T stage, tumor budding, and microsatellite instability-negative phenotype. Among the 71 patients with high T stage and microsatellite instability-negative phenotype, tumors with isolated tumor cells were characterized by a high frequency of tumor budding compared with tumors without isolated tumor cells (85 vs. 36.4 percent). In a further study, the degree of budding, which was assessed by an immunohistochemical study of 2 chain of laminin-5, was closely related to the number and location of isolated tumor cells. Moreover, we found that most of the isolated tumor cells in the regional lymph nodes also expressed 2 chain of laminin-5.CONCLUSIONS Our results suggested that isolated tumor cells are derived from undifferentiated cancer cells or small clusters (budding) at the invasive front. Thus, tumor budding may be used as an indicator of isolated tumor cells in lymph nodes with node-negative colorectal cancers.Supported by a grant from the Seoul National University Bundang Hospital Research Fund, Seongnam, Korea.Presented at the meeting of The Korean Society of Pathologists, Seoul, Korea, October 30 to November 1, 2003.     

Early and multiple origins of metastatic lineages within primary tumors

Many aspects of the evolutionary process of tumorigenesis that are fundamental to cancer biology and targeted treatment have been challenging to reveal, such as the divergence times and genetic clonality of metastatic lineages. To address these challenges, we performed tumor phylogenetics using molecular evolutionary models, reconstructed ancestral states of somatic mutations, and inferred cancer chronograms to yield three conclusions. First, in contrast to a linear model of cancer progression, metastases can originate from divergent lineages within primary tumors. Evolved genetic changes in cancer lineages likely affect only the proclivity toward metastasis. Single genetic changes are unlikely to be necessary or sufficient for metastasis. Second, metastatic lineages can arise early in tumor development, sometimes long before diagnosis. The early genetic divergence of some metastatic lineages directs attention toward research on driver genes that are mutated early in cancer evolution. Last, the temporal order of occurrence of driver mutations can be inferred from phylogenetic analysis of cancer chronograms, guiding development of targeted therapeutics effective against primary tumors and metastases.It has long been understood that tumorigenesis is an evolutionary process (1) associated with the accumulation of somatic mutations (2). However, many aspects of that process that are fundamental to cancer biology and targeted treatment have been challenging to reveal, such as the divergence times and genetic clonality of metastatic lineages (3, 4). Somatic mutations have revealed tumor type-specific drivers by comparison of primary tumor and normal tissues (5, 6), and studies examining the evolutionary process of cancer across multiple sites have used a handful of subjects to identify ubiquitous, shared, and private mutations (1) and to reconstruct a number of tumor phylogenies using parsimony or unweighted pair group methods with arithmetic mean (1, 7) but have lacked the power to generalize about the tumorigenic or metastatic process across cancer types (1).Tumor phylogenetics, using a larger sample with explicit evolutionary models, can be applied using molecular evolutionary models to reconstruct ancestral states of somatic mutations and infer cancer chronograms, revealing novel information about the timing of gene mutations and their contributions to tumorigenesis and metastasis and addressing three fundamental aspects of cancer biology. First, the topology of divergence of primary and metastatic lineages can differentiate between a linear model of cancer progression, in which all metastatic tumors are descended from a single original primary cell such that all metastases are more closely related to each other than they are to any tissue in the primary tumor, and a branched model, in which metastases can originate from divergent lineages within primary tumors. Second, molecular evolutionary trees and chronograms can quantify how early metastatic lineages arise in tumor development, clarifying the role of mutations in facilitating metastasis. Last, integration of temporal inferences across patients can convey the order of occurrence of driver mutations, guiding development of targeted therapeutics effective against primary tumors and metastases.Here, we perform tumor phylogenetics to address these questions. Although ascertaining variable degrees of tumor heterogeneity (1) by computational analyses of subclonality within primary tumors has proven challenging (8), another approach to revealing heterogeneity is analysis of the sequence divergence of major clones extracted from distant sites. We replayed the “tape of cancer” and mapped genetic mutations on the tree of cancer evolution extending from normal tissue to primary tumor and metastases. Analyzing new exome sequence data from primary and metastatic sites, we applied maximum likelihood and Bayesian approaches to reveal phylogenetic relationships and tumor evolution chronology. We identified genetic mutations associated with tumorigenesis that commonly precede the first genetic divergence of all cancer lineages, also examining those that precede all metastases. Furthermore, we quantified the temporal distributions of the first genetic divergence of metastases from the primary tumor and evaluated the temporal order of gene mutations in cancer.     

Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.During metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed “tumor clumps”) are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5–7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of epithelial tumors, a process termed “collective invasion” (9, 10). In breast cancer, collective invasion is facilitated by invasive leader cells, a subpopulation of tumor cells that highly express keratin 14 (K14) and other basal epithelial markers (11). K14⁺ cells are migratory, protrusive, and lead trailing K14⁻ cells, while maintaining cell–cell cohesion and E-cadherin–based cell contacts.In this study, we sought to understand how these K14⁺ cells exit collective invasion strands in the primary tumor and travel to distant organs (12). One hypothesis is that collective invasion is an intermediate step toward eventual single-cell dissemination and monoclonal metastasis. However, tumor cell clusters are detected in circulation (5) and primary human breast tumors can disseminate collectively into the surrounding extracellular matrix in ex vivo assays (13–15). These data prompted an alternative hypothesis, that collectively invading K14⁺ cancer cells could initiate and complete the metastatic process as a cohesive multicellular unit. Here we define the clonal nature of metastases in a spontaneous mouse model of metastasis to the lungs (16, 17), in which the predominant invasive form in the primary tumor is collective invasion strands led by K14⁺ cells (11). We establish that the majority of metastases arise from polyclonal seeds, and show that disseminated tumor cell clusters are predominantly composed of K14⁺ cells. We propose a mechanism for polyclonal metastasis via the collective invasion, dissemination, and colonization of clusters of K14⁺ cancer cells.     

Bone metastases in germ cell tumor patients

Purpose

Little data exist on characteristics, treatment, and outcome of patients with bone metastases from germ cell cancer.

Methods

A total of 434 patients with poor prognosis germ cell cancer, who underwent primary high-dose chemotherapy (HD-CTX) within two phase II trials, were retrospectively analyzed.

Results

40 patients (9%) presented with primary bone metastases. Bone metastases were significantly more frequently observed in patients with primary mediastinal tumors, yolk sac tumor histology, and synchronous liver metastases. Overall response rate to HD-CTX was 85%. 20% of patients underwent consolidating radiotherapy, and 10% had resection of bone metastases revealing necrosis in all cases. Progression-free survival rate after primary treatment was 63% and, including salvage treatment after first relapse, overall long-term survival rate was 75%. Four patients (0.9%) relapsed with isolated bone metastases, all with bone metastases at primary diagnosis. None had previously received surgery or radiotherapy and all died within 1?year. 10 patients with primary bone metastases showed recurrences at other localizations. No patient relapsed with bone plus other metastases or with de novo bone metastases.

Conclusions

Bone metastases were associated with a primary mediastinal nonseminoma, yolk sac histology, and liver metastases at first diagnosis. In this cohort of patients receiving HD-CTX as first-line treatment, 63% achieved long-term progression-free survival. Skeletal relapses were rare, but showed dismal outcome.