Microscopic Metastases Research Articles

LION: Lymphadenectomy in ovarian neoplasms—A prospective randomized AGO study group led gynecologic cancer intergroup trial.

5500 Background: So far, there is no level-1 evidence regarding the role of systematic pelvic and para-aortic lymphadenectomy (LNE) in patients with advanced ovarian cancer (AOC) with macroscopic complete resection und clinically negative lymph nodes (LN). Therefore, surgical management regarding LNE worldwide is very heterogeneous. Methods: Prospective randomized trial including patients with newly diagnosed AOC FIGO IIB-IV with macroscopic complete resection and pre- and intra-operatively clinical negative LN were randomized intra-operatively to LNE versus no-LNE. All centers had to qualify regarding surgical skills before participation in this trial. The primary endpoint was overall survival. Results: 647 patients were randomized between 12/08 and 1/12 to LNE (n=323) or no-LNE (n=324). The median number of removed LN in patients randomized to LNE was 57 (pelvic 35 and para-aortic22). Post-op platinum-taxane based chemotherapy was applied in 85% of the patients in the no-LNE arm and 80% in the LNE arm. Microscopic metastases were diagnosed in 56% of the pts in the LNE arm. Median OS in the no-LNE arm was 69 months and 66 months in the LNE arm (HR 1.06, 95%CI 0.83-1.34, p=0.65) and the median PFS was 26 months in both arms (HR 1.11, 95%CI 0.92-1.34 p=0.30). Surgery in the LNE arm was 64 minutes longer (means: 352 vs 288 min), resulted in a higher median blood loss (650 vs 500 ml), and a higher transfusion rate (67% vs 59%). Furthermore, serious post-operative complications occurred more frequently in the LNE arm (e.g. rate of re-laparotomies 12.1% vs 5.9% [p=0.006], hospital re-admittance rate 8.0% vs 3.1% [p=0.006] and deaths within 60 days after surgery 3.1 vs 0.9% [p=0.049]). Conclusions: Systematic pelvic and para-aortic LNE in patients with AOC with both intra-abdominal complete resection and clinically negative LN neither improve overall nor progression-free survival despite detecting (and removing) sub-clinical retroperitoneal lymph node metastases in 56% of the patients. Our data indicate that systematic LNE of clinical negative LN in patients with AOC and complete resection should be omitted to reduce post-operative morbidity and mortality. Clinical trial information: NCT00712218.

Bone-in-culture array as a platform to model early-stage bone metastases and discover anti-metastasis therapies

The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases.

Abstract P4-06-11: Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts

Abstract Background: Generation of BT-474 and MDA-MB-231 xenografts in immunocompromised mice provides one means of dissecting the molecular events involved in local invasion versus distant metastasis. Recently, Glunde et al have shown an interdependence of genes involved in cell-cell and cell-matrix adhesion (E-cadherin and integrin β1) and extra-cellular matrix degradation (MMP-2 and 9) in allowing cells to metastasize. Further, Fite el al have identified a set of microRNAs (miRs) up-regulated by E-cadherin (a pre-EMT marker) and down-regulated by Vimentin (post-EMT marker) in acquiring an invasive phenotype. We have performed a detailed analysis of integrins, matrix metallo-proteases and key miRs to better understand the molecular events underlying these disparate behaviours. Methods: We injected BT-474 (N=5) and MDA-MB-231 (N=5) cells orthotopically into SCID mice. Xenografts were assessed for local growth rate and monitored for distant metastasis. The implanted tumors as well as the distant metastatic foci were harvested. Markers involved in local invasion, distant metastasis and tumor-stroma interactions including miRs were compared between BT-474 and MDA-MB-231 cell lines and their xenografts by q-RT-PCR, immunofluorescence and immunohistochemistry. Results: As expected BT-474 xenografts showed a higher rate of tumor growth when compared to MDA-MB-231. Histological examination of BT-474 tumors confirmed only locally invasive tumor growth with infiltrated blood capillaries and vessels; no macro and microscopic metastases were observed in the organs collected. On the contrary, MDA-MB-231 xenografts showed highly undifferentiated tumor growth and frank lung metastasis and extra-pulmonary tumor growth in one of the five mice injected despite slow rate of local growth. Expression of matrix metalloproteases – MMP-2 & 9 was more than 30 fold upregulated in MDA-MB-231 xenografts as compared to BT-474. Elevated level of E-cadherin was observed in BT-474 but was absent in MDA-MB-231. The most interesting differences were seen in the levels of miRs and cell-surface integrins. High levels of miR-18a, miR-93 and miR-182 were observed in BT-474 implants when compared to MDA-MB-231 which had a much lower level of these miRs. On the contrary, higher levels of integrin β3, and β1 were observed in MDA-MB-231 tumors when compared to BT-474. Integrin β6 was absent in both. The reciprocal relationship between these markers is being examined and compared between locally invasive tumors and metastatic triple negative breast cancers from our case series of human specimens (N=250). Conclusion: miRs and integrins known to be involved in invasion are differentially regulated in tumors that are locally invasive compared to ones with distant metastasis. The level of the key targets of these miRs as well as additional integrins is being examined. Understanding the epigenetic regulations leading to metastasis via tumor-stroma interaction might help in discerning differential tumor behaviour. Citation Format: Lawrence PV, Desai K, Prabhu JS, Korlimarla A, Nair MG, Sridhar TS. Differential regulation of microRNAs and integrins influences metastatic potential: Comparison between locally invasive BT-474 and metastatic MDA-MB-231 xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-11.

SC02.03 Surgery for Ground Glass Opacity: Sublobar Resection?

SC02.03 Surgery for Ground Glass Opacity: Sublobar Resection?

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Abstract 1525: Vortex technology for label-free enrichment of CTC from mouse xenograft models

Abstract Background Non-invasive liquid biopsies, such as CTCs, have been of growing interest due to their potential use in cancer detection, prognosis, and monitoring of therapeutic resistance [1]. Beyond enumeration, characterization of CTCs could help guide treatment selection and the development of targeted cancer therapy. Here, we show that Vortex technology can be used successfully for the size-based capture of CTCs in a preclinical mouse model of breast cancer. Method To establish that human epithelial cancer cells can be reliably detected in small volumes of mice blood, 50-100 MDA-MB231 and MCF7 breast cancer cells were spiked in 500 μL mice blood, diluted 20 fold, and processed through Vortex chip [2]. Recovered cells were immunostained (CK, CD45, DAPI), and enumerated. For breast cancer xenograft model, 8×106 MDA-MB-231-fLuc/GFP cells were implanted orthotopically into the mammary fat pad of NOD-SCID Gamma mice (n = 35). Tumors were measured in 2 dimensions 3 times/week and tumor volume calculated. Blood from cardiac puncture (500 μl) and lateral saphenous vein (100 μl) was collected starting 1 week post implantation, diluted 40X and processed. Mice were euthanized, organs harvested, formalin fixed, paraffin embedded and H&E stained. Results Spiking of MCF7 cells in mouse blood resulted in a capture efficiency of 56.9±13% and a purity of 80.1± 9% (n = 3), with the flow-through from each cycle being re-processed for a total of 4 cycles. Recycling the flow-through led to capture efficiency of 36.7%, 23.4%, 5.2% and 7.9% during cycles 1, 2, 3 and 4 respectively. Similar results were obtained for MDA-MB-231, with a capture efficiency of 54.4% and a purity of 41.3%. In an orthotopic tumor xenograft model of breast cancer, CTCs were successfully recovered from cardiac puncture as early as day 7. CTC counts, ranging from 0.4-3649 CTCs/100 μl, increased over time and correlated with tumor burden. CTC clusters were also captured from day 7 (6 clusters/100 μl; frequency 1/3), with number and frequency increasing over time up to 147-485 clusters/100 μl by day 42. No CTC were recovered from lateral saphenous vein blood until day 28 post implantation, and their number remained low (mean 2.15±0.65). Microscopic metastases were evident in lung of all mice starting at day 28 and in liver of all mice starting at day 35. Conclusion CTCs were isolated in a label-free manner from mice blood with both high capture efficiency and purity. In a preclinical model of metastatic breast cancer, CTC counts correlated well with primary tumor volume and metastases occurrence. Thus the Vortex chip appears to be well suited for the enrichment of CTCs from murine xenograft models. Future works will focus on mice implanted with patient derived xenograft (PDX) and their therapeutic response. Information gathered from these studies should facilitate discovery of new therapeutic targets and the development of personalized medicine. [1] Ignatiadis et al. Clin Cancer Res 2015. [2] Sollier et al. Lab Chip 2014.[K.H. and M.T. contributed equally to this work.] Citation Format: Kyra Heirich, Melanie M. Triboulet, Corinne M. Renier, Vishnu C. Ramani, Elodie Sollier, Stefanie S. Jeffrey. Vortex technology for label-free enrichment of CTC from mouse xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1525.

Abstract 1579: Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells

Abstract Purpose: The peritoneum and organs in the peritoneal cavity are covered by a single layer of mesothelial cells. Therefore, ovarian cancer cells, which metastasize within the peritoneal cavity, directly encounter mesothelial cells as the initial step of metastasis. This contact has been found to involve cell-cell communication that affects cancer progression. Possible actors in this cell-cell communication are exosomes, 30-100 nm membrane vesicles of endocytic origin, through the cell-cell transfer of proteins, mRNAs, and microRNAs. Here, we aim to identify the functional roles of ovarian cancer-derived exosomes in this metastatic process. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope, nanoparticle tracking analysis, Western blotting and electrophoresis of RNA. The transfer of exosomes into HPMCs was confirmed by fluorescent-labeled exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells in cancer invasion were analyzed in vitro 3D culture model, morphological assessment, Western blotting and gelatin zymography. CD-44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD-44 was analyzed. CD-44 expression in ovarian cancer omental metastasis and surrounding organs was assessed by immunohistochemistry using clinical samples. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. CD-44 was enriched in cancer-derived exosomes and exosome-treated HPMCs display high-level of CD-44. When CD-44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD-44 into HPMCs promoted cancer invasion. In human omentum with microscopic metastasis of ovarian cancer, positive CD-44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD-44 expression was generally negative in normal mesothelial cells. Conclusion: Ovarian cancer-derived exosomes transfer CD-44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing mesothelial clearance. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosomes promote ovarian cancer invasion through CD-44 transfer to mesothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1579.

Abstract A82: Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization.

Abstract Introduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures. Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6-/-); T cells (Nude); B and T cells (Rag1-/-); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc. Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b+ milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies. Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth. New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b+ cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media. To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b+ cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media. In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing. Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number. Citation Format: Venkatesh Krishnan, Kelly Mitchell, Jason Miska, Sophia George, Patricia Shaw, Victoria Seewaldt, Maciej Lesniak, Marina Chekmareva, Lev Becker, Vinita Parkash, Cindy Miranti, Carrie Rinker-Schaeffer. Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A82.

Antitumor Effect of IP-10 by Using Two Different Approaches: Live Delivery System and Gene Therapy.

PurposeImmunotherapy is one of the treatment strategies for breast cancer, the most common cancer in women worldwide. In this approach, the patient's immune system is stimulated to attack microscopic tumors and control metastasis. Here, we used interferon γ-induced protein 10 (IP-10), which induces and strengthens antitumor immunity, as an immunotherapeutic agent. We employed Leishmania tarentolae, a nonpathogenic lizard parasite that lacks the ability to persist in mammalian macrophages, was used as a live delivery system for carrying the immunotherapeutic agent. It has been already shown that arginase activity, and consequently, polyamine production, are associated with tumor progression.MethodsA live delivery system was constructed by stable transfection of pLEXSY plasmid containing the IP-10-enhanced green fluorescent protein (IP-10-egfp) fusion gene into L. tarentolae. Then, the presence of the IP-10-egfp gene and the accurate integration location into the parasite genome were confirmed. The therapeutic efficacy of IP-10 delivered via L. tarentolae and recombinant pcDNA-(IP-10-egfp) plasmid was compared by determining the arginase activity in a mouse 4T1 breast cancer model.ResultsThe pcDNA-(IP-10-egfp) group showed a significant reduction in tumor weight and growth. Histological evaluation also revealed that only this group demonstrated inhibition of metastasis to the lung tissue. The arginase activity in the tissue of the pcDNA-(IP-10-egfp) mice significantly decreased in comparison with that in normal mice. No significant difference was observed in arginase activity in the sera of mice receiving other therapeutic strategies.ConclusionOur data indicates that IP-10 immunotherapy is a promising strategy for breast cancer treatment, as shown in the 4T1-implanted BALB/c mouse model. However, the L. tarentolae-(IP-10-EGFP) live delivery system requires dose modifications to achieve efficacy in the applied regimen (six injections in 3 weeks). Our results indicate that the arginase assay could be a good biomarker to differentiate tumoral tissues from the normal ones.

Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior.

ABSTRACTOsteosarcoma (OS) is a heterogeneous and rare disease with a disproportionate impact because it mainly affects children and adolescents. Lamentably, more than half of patients with OS succumb to metastatic disease. Clarification of the etiology of the disease, development of better strategies to manage progression, and methods to guide personalized treatments are among the unmet health needs for OS patients. Progress in managing the disease has been hindered by the extreme heterogeneity of OS; thus, better models that accurately recapitulate the natural heterogeneity of the disease are needed. For this study, we used cell lines derived from two spontaneous canine OS tumors with distinctly different biological behavior (OS-1 and OS-2) for heterotypic in vivo modeling that recapitulates the heterogeneous biology and behavior of this disease. Both cell lines demonstrated stability of the transcriptome when grown as orthotopic xenografts in athymic nude mice. Consistent with the behavior of the original tumors, OS-2 xenografts grew more rapidly at the primary site and had greater propensity to disseminate to lung and establish microscopic metastasis. Moreover, OS-2 promoted formation of a different tumor-associated stromal environment than OS-1 xenografts. OS-2-derived tumors comprised a larger percentage of the xenograft tumors than OS-1-derived tumors. In addition, a robust pro-inflammatory population dominated the stromal cell infiltrates in OS-2 xenografts, whereas a mesenchymal population with a gene signature reflecting myogenic signaling dominated those in the OS-1 xenografts. Our studies show that canine OS cell lines maintain intrinsic features of the tumors from which they were derived and recapitulate the heterogeneous biology and behavior of bone cancer in mouse models. This system provides a resource to understand essential interactions between tumor cells and the stromal environment that drive the progression and metastatic propensity of OS.

The meaning of gross tumor type in the aspects of cytokeratin 19 expression and resection margin in patients with hepatocellular carcinoma.

There is no consensus on the safe resection margin in patients with hepatocellular carcinoma. Surgeons decide the extent of resection according to residual liver function and tumor location. We investigated the influence of resection margin on early recurrence with respect to gross tumor type and expression of cytokeratin 19 (CK19). We divided the patients into two groups based on the classification of The Korean Liver Cancer Study group as follows: group 1 included expanding and vaguely nodular types whereas group 2 included nodular with perinodular extension, multinodular confluent, and infiltrative types. We classified the resection margin as narrow (0.1-0.9 cm) or wide (greater than 1 cm). We compared clinicopathological features and CK19 positivity between the groups. Group 2 had a higher prevalence of gross portal vein invasion, microscopic portal vein invasion, microvessel invasion, satellite nodules, intrahepatic metastasis, multicentric occurrence, and positivity for CK19. Group 1 showed no difference in recurrence according to the resection margin; however, group 2 showed a higher recurrence rate in patients with a narrow resection margin compared with those with a wide resection margin (P = 0.047). Patients in group 2 with CK19 positivity showed a higher prevalence of microvessel invasion than those without CK19 (P = 0.026). Although our study has the limitation of a small number of cases, the data suggest that patients with hepatocellular carcinoma of expanding and vaguely nodular gross types may safely undergo surgical resection with a narrow resection margin and a low risk of early recurrence.

Abstract IA22: Preclinical modeling of adjuvant and metastatic antiangiogenic therapy: Relevance for better predicting clinical outcomes

Abstract Over the last decade my lab has developed a number of models involving treatment of mice with either early stage microscopic metastatic disease for adjuvant therapy or late stage overt metastatic disease for metastatic therapy studies1-3. More recently, models of neoadjuvant therapy have been developed as well with the lab of Dr. John Ebos4. The rationale for utilizing the first two models is that they may be superior in predicting future activity in patients enrolled in randomized phase III adjuvant or metastatic therapy clinical trials, in comparison to conventional treatment models involving mice with unresected established primary tumors, and evaluating the effect on the primary tumor growth only1. As an example, we reported that sunitinib (or pazopanib) or DC101, the VEGFR-2 antibody were all devoid of anti-tumor activity when treating mice with advanced metastatic breast cancer after primary tumor resection, whereas in contrast, all showed efficacy when treating orthotopic primary tumors in control experiments5. Combining chemotherapy with sunitinib also did not improve outcomes in the metastatic setting. In contrast, combining chemotherapy with DC101 caused a small but statistically significant benefit in survival. These results retrospectively correlated with outcomes of four metastatic breast cancer phase III trials evaluating sunitinib alone or in combination with chemotherapy in the metastatic setting (all were negative) or multiple phase III trials evaluating the VEGF antibody, bevacizumab, with chemotherapy, which showed variable benefits in improving PFS in metastatic breast cancer5. With respect to adjuvant therapy modelling, we reported in 2009 that adjuvant sunitinib therapy of mice with microscopic metastases after resection of orthotopic primary human breast cancer xenografts resulted in a worsened survival outcome, with accelerated progression of metastatic disease2. On the basis of the results we raised a cautionary “flag” about the rationale of antiangiogenic drugs in the clinic for adjuvant setting6. As is now well known, there have been multiple adjuvant trials evaluating bevacizumab plus chemotherapy in postsurgical early stage colorectal or breast cancer, as well as sorafenib in hepatocellular carcinoma, and all of these trials have failed to meet their primary endpoint of a benefit in disease free survival7. This has provoked considerable discussion and debate about the basis for such failures in contrast to the same drugs/therapies showing efficacy in the more advanced metastatic settings of the same malignancies. One basis for the modest positive effects noted of such therapies in the metastatic setting, and for the failure in the adjuvant setting, concerns the impact that “vessel co-option”, especially in distant metastases, may have on therapeutic outcomes. Evidence is growing that a variety of tumors and especially overt metastases in certain sites such as the lungs, liver, and brain are minimally or non-angiogenic and instead “hijack” the existing vasculature in such organ sites8. The same may be the case for microscopic metastases. Consequently, there will be growing interest in evaluating whether vessel co-option can be therapeutically targeted (and also what the implications may be for drug-induced vascular normalization). In this regard there are a number of strategies being evaluated such as the impact of metronomic chemotherapy and targeting other pro-angiogenic factors/pathways beyond VEGF such as ang2/tie29, which may be effective as an adjuvant therapy strategy9.

Sentinel European Node Trial (SENT): 3-year results of sentinel node biopsy in oral cancer

PurposeOptimum management of the N0 neck is unresolved in oral cancer. Sentinel node biopsy (SNB) can reliably detect microscopic lymph node metastasis. The object of this study was to establish whether the technique was both reliable in staging the N0 neck and a safe oncological procedure in patients with early-stage oral squamous cell carcinoma. MethodsAn European Organisation for Research and Treatment of Cancer-approved prospective, observational study commenced in 2005. Fourteen European centres recruited 415 patients with radiologically staged T1–T2N0 squamous cell carcinoma. SNB was undertaken with an average of 3.2 nodes removed per patient. Patients were excluded if the sentinel node (SN) could not be identified. A positive SN led to a neck dissection within 3 weeks. Analysis was performed at 3-year follow-up. ResultsAn SN was found in 99.5% of cases. Positive SNs were found in 23% (94 in 415). A false-negative result occurred in 14% (15 in 109) of patients, of whom eight were subsequently rescued by salvage therapy. Recurrence after a positive SNB and subsequent neck dissection occurred in 22 patients, of which 16 (73%) were in the neck and just six patients were rescued. Only minor complications (3%) were reported following SNB. Disease-specific survival was 94%. The sensitivity of SNB was 86% and the negative predictive value 95%. ConclusionThese data show that SNB is a reliable and safe oncological technique for staging the clinically N0 neck in patients with T1 and T2 oral cancer.EORTC Protocol 24021: Sentinel Node Biopsy in the Management of Oral and Oropharyngeal Squamous Cell Carcinoma.

Locoregional failure rates after mastectomy for breast cancer patients with T1-2 tumors and axillary nodal microscopic metastases.

64 Background: The indications for postmastectomy radiotherapy (PMRT) are expanding to include patients 1-3 axillary nodal metastases (ALN). Improvements in diagnostic evaluation have led to increasing numbers of breast cancer (BC) patients who are found to have microscopic nodal metastases (N1mic). The challenge today is whether these BC patients have risk that warrants the routine delivery of PMRT. Methods: We reviewed patients with pathologic T1-2N1 BC treated with initial mastectomy (mast) and adjuvant systemic therapy (ST) from 2000-2013. The primary endpoint was locoregional failure (LRF), defined as a recurrence in either the ipsilateral chestwall or regional lymphatics (axillary, internal mammary, or supraclavicular). Secondary endpoints were disease-free survival (DFS, failure or death) and overall survival (OS). The log-rank test was used to compare survival between groups. Results: We identified 550 eligible patients from our prospectively maintained cancer registry with 5 year median follow-up. 95 patients (17%) had N1mic disease. Baseline characteristics include: median age 53 yrs, 61% pathologic T2, 39% grade 3, 72% hormone receptor positive, 16% HER2+, 12% triple-negative. Treatment included chemotherapy in 78% (n = 428), PMRT in 15% (n = 82), and anti-endocrine therapy in 70% (n = 385). A median of 18 ALN (range, 1-68) were removed. Among the patients with N1mic disease, 81 had 1+ node, 13 had 2+ nodes, and 1 had 3+ nodes. The 5 yr LRF was 0% for patients with N1mic disease vs. 4.6% in those macro metastases (p = 0.84). The 5 yr LRF rate for the entire cohort was 3.9%; patients with 1+, 2+, and 3+ nodes had 5 yr LRF of 2.6%, 4.7% and 6.4%, respectively (p = 0.79). Patients with N1mic disease had a trend towards improved DFS (91.6% vs. 82.3%, p = 0.07) and significantly improved OS (96.9% vs. 87.6%, p = 0.03) compared to patients with macrometastases. Conclusions: In a cohort of patients with T1-2,N1 BC treated with modern therapy, we found overall low rates of LRF. Patients with N1mic disease had no LRF events and improved OS compared to patients with macrometastases. These findings support that PMRT should not be routinely recommended for N1mic BC patients with T1-2 tumors.

Abstract 3370: Incorporation of mesenchymal stem cells into areas of lung metastasis in an osteosarcoma model

Abstract Lung is the most common metastatic site in osteosarcoma patients with a majority presenting lung micrometastasis at diagnosis time. MSCs compose a heterogeneous cell population with the ability to migrate and home into remodeling tissues, including tumors. To evaluate the participation of MSCs in lung metastasis formation, we intravenously injected LM7 osteosarcoma cells into immunosuppressed mice. Pre-labelled MSCs (DiR+/CMDiI+, at a dose of 0.5-1×10e6 cells) were intravenously administered 10 weeks after tumor cells injection, when microscopic metastasis become apparent. MSCs biodistribution was evaluated both through real time (DiR+) and microscopic (CMDiI+) analysis. MSCs levels in the lungs of tumor-bearing mice as compared to mice with no tumor burden was 1.15×10E5±0.07 p2/sec/cm2/sr vs. 5.44×10E4±0.5p2/sec/cm2/sr (one week post-inoculation, infrared signal, p<0.01). This indicates a high homing ability of the stromal cells into the metastatic tumor microenvironment. Microscopic analysis corroborated infrared signal with MSCs presence and incorporation within the metastatic sites. In addition, MSCs displayed heterogeneous distribution within lungs with LM7 metastasis as opposed to the homogeneous MSCs distribution observed in lungs without metastasis. This suggests that MSCs are chemoattracted and retained in preferential areas within the lungs with LM7 osteosarcoma metastases. The setup of an experimental osteosarcoma metastatic model which incorporates administered MSCs as tumor stromal cells will be useful to analyze MSCs function in tumor metastasis formation. Note: This abstract was not presented at the meeting. Citation Format: Luciana M. Gutierrez, Mariana A. Amoros, Gustavo Sevlever, Osvaldo Podahjcer, Graciela Cremaschi, Eugenie S. Kleinerman, Marcela F. Bolontrade. Incorporation of mesenchymal stem cells into areas of lung metastasis in an osteosarcoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2015-3370

Abstract 5060: Exosome transfer from ovarian cancer cells to mesothelial cells promotes cell invasion by upregulating MMP-9 secretion and increasing clearance of mesothelial cells

Abstract Ovarian cancer cells metastasize to organs in the abdominal cavity, such as the omentum, which are covered by a single layer of mesothelial cells. Thus, the cell-cell connection between ovarian cancer cells and mesothelial cells is the crucial step of metastasis. Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions through transfer of proteins, mRNAs and microRNAs. However, whether exosome-mediated transfer plays any role in ovarian cancer cell invasion remains poorly understood. Thus, the aim of this study is to identify the functional role of ovarian cancer-derived exosomes during the process of ovarian cancer metastasis. Methods: Exosomes were isolated from two ovarian cancer cell lines (HeyA8 and TYK-NU) and immortalized normal ovarian epithelial cell line (IOSE) using differential centrifugation. Human peritoneal mesothelial cells (HPMCs) were isolated from normal omentum of patients undergoing gynecologic surgery. The isolation of exosomes was confirmed by electron microscope and the transfer of exosomes into HPMCs was confirmed by fluorescent-labeling exosomes. The effects of exosome transfer from ovarian cancer cells to mesothelial cells were analyzed in vitro 3D culture model, morphological assessment, Western Blotting and gelatin zymography. We found that CD44 was enriched in cancer derived exosome. Thus, gain or loss of function of CD44 was analyzed. CD44 expression of ovarian cancer omental metastasis and surrounding organs in clinical samples was assessed by immunohistochemistry. Results: Fluorescent-labeled exosomes were evidently transferred into HPMCs. Exosome-treated HPMCs changed in cellular morphology to spindle phenotype. Ovarian cancer invasion was significantly promoted in the presence of exosome-treated HPMCs. In exosome-treated HPMCs, MMP-9 secretion was up-regulated and E-cadherin expression was down-regulated. The clearance of mesothelial barrier was increased in exosome-treated HPMC monolayer. By Western Blotting, we confirmed CD44 was enriched in exosomes and exosome-treated HPMCs display high-level of CD44. When CD44 expression was knocked down by siRNA in ovarian cancer cells, these effects to HPMCs were significantly attenuated. In contrast, the enforced expression of CD44 into HPMCs promoted cancer invasion by secreting MMP-9 and increasing mesothelial clearance. In human omentum with microscopic metastasis of ovarian cancer, positive CD44 expression was confirmed in a mesothelial cell layer when cancer cells are attaching onto it, while CD44 expression was generally negative in normal mesothelial cells. Conclusion: Herein, we revealed that ovarian cancer-derived exosomes transfer CD44 to HPMCs, which can facilitate ovarian cancer invasion by up-regulating of MMP-9 secretion and increasing clearance of HPMCs. Citation Format: Koji Nakamura, Kenjiro Sawada, Yasuto Kinose, Akihiko Yoshimura, Erika Nakatsuka, Seiji Mabuchi, Tadashi Kimura. Exosome transfer from ovarian cancer cells to mesothelial cells promotes cell invasion by upregulating MMP-9 secretion and increasing clearance of mesothelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5060. doi:10.1158/1538-7445.AM2015-5060

Ovarian involvement in endometrioid adenocarcinoma of uterus

ObjectiveOvarian preservation is an option for some premenopausal patients with early stage endometrial cancer. Studies have shown that ovarian preservation in selected patients does not negatively impact survival outcomes. The objective of this study is to determine the frequency and characteristics of ovarian involvement when endometrial cancer is clinically confined to the uterus. MethodsPatients with endometrioid adenocarcinoma of uterus treated at our institution between 2000 and 2013 were identified. Patients with ovarian metastasis or synchronous primary ovarian cancer were included. Patients were excluded if there was gross extrapelvic disease on examination or imaging. ResultsSeven hundred and fifty-nine patients were found to have endometrial cancer with the disease confined to the pelvis (stages I, II, and III). Fifteen patients (2%) had ovarian metastasis. Twenty-three patients (3%) had synchronous uterine and ovarian cancer. Most ovarian lesions (32 out of 38) were either enlarged or had abnormal appearing surface involvement. Six patients had microscopic ovarian involvement, accounting for 0.8% of the endometrial cancer patients with pelvis-confined disease. All of the patients were greater than 50years of age. For those patients with microscopic ovarian metastasis, all had FIGO grade 3 disease, deep myometrial invasion, and extrauterine involvement of either cervix or lymph nodes. ConclusionsMicroscopic ovarian involvement occurred in 0.8% of patients with endometrial cancer. For premenopausal patients with endometrial cancer, normal appearing ovaries may be considered for preservation in the absence of extrauterine spread, grade 3 disease and deep myometrial invasion.

Shoulder Dysfunction after Selective Neck Dissection in Recurrent Nasopharyngeal Carcinoma.

To investigate the incidence of occult nodal metastasis and severity of shoulder dysfunction after selective neck dissection (SND) for recurrent nasopharyngeal carcinoma (NPC) with N0 status. Prospective, single-group, pre/post test design. Academic medical center. Between 1998 and 2012, 46 patients who had recurrent NPC and N0 status were recruited. They subsequently received salvage nasopharyngectomy and SND, removing ipsilateral level I to III and V lymphatics. The incidence of occult nodal metastasis was noted. All patients underwent standardized physiotherapy after surgery. Postoperative shoulder function was measured using the Disability of Arm, Shoulder, and Hand (DASH) questionnaire. The incidence of microscopic nodal metastasis was 15.2%. For first year posttreatment, the mean DASH score was 44.2. With time, there was no improvement in shoulder function despite targeted physiotherapy (P = .09), and the second postoperative year mean DASH score was 46.3. The degree of daily activity affected was rated as moderate to very limited, and 30% of the patients had at least moderate shoulder pain at rest. Shoulder dysfunction after SND for recurrent NPC is significant and persistent. Given the low incidence of microscopic nodal metastasis in such circumstances, routine SND is not recommended.

Feasibility, Sensitivity, and Specificity of Postprocedure Peritoneal Cytology.

The objective of this study was to determine the feasibility and diagnostic performance of cytopathologic evaluation of postprocedure washings collected after hysterectomy for gynecologic cancer. A total of 92 cases of hysterectomy for malignancy having cytology reports of both pre- and postprocedure washings were retrospectively identified. In all, 98.7% of preprocedure and 99.3% of postprocedure washings (P = 1.00) were satisfactory for cytopathology. Discordance regarding the observation of malignant cells between preprocedure and postprocedure washings was insignificant (P = .267). The sensitivity of postprocedure cytology for detecting malignant cells in cases of positive peritoneal histology was significantly lower than the sensitivity of preprocedure cytology (28.6% vs 57.1%, P = .041), with similar specificities (both 94%). Four patients with endometrial cancer having negative preprocedure peritoneal cytology were discovered to have positive postprocedure cytology. Postprocedure peritoneal cytology is feasible and may benefit patients with early-stage cancer by increasing the detection of microscopic peritoneal metastasis or cancer cell seeding during surgery.