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PURPOSE In this phase I study (BLOOM), osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was evaluated in patients with leptomeningeal metastases (LMs) from EGFR-mutated (EGFRm) advanced non–small-cell lung cancer (NSCLC) whose disease had progressed on previous EGFR-TKI therapy. PATIENTS AND METHODS Patients with cytologically confirmed LM received osimertinib 160 mg once daily. Objectives were to assess confirmed objective response rate (ORR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), pharmacokinetics (PK), and safety. Additional efficacy evaluations included changes from baseline in CSF cytology and neurologic examination. Measurable lesions were assessed by investigator according to RECIST version 1.1. LMs were assessed by neuroradiologic blinded central independent review (BICR) according to Response Assessment in Neuro-Oncology LM radiologic criteria and by investigator. RESULTS Forty-one patients were enrolled. LM ORR and DoR by neuroradiologic BICR were 62% (95% CI, 45% to 78%) and 15.2 months (95% CI, 7.5 to 17.5 months), respectively. Overall, ORR by investigator was 41% (95% CI, 26% to 58%), and median DoR was 8.3 months (95% CI, 5.6 to 16.5 months). Median investigator-assessed PFS was 8.6 months (95% CI, 5.4 to 13.7 months) with 78% maturity; median OS was 11.0 months (95% CI, 8.0 to 18.0 months) with 68% maturity. CSF tumor cell clearance was confirmed in 11 (28%; 95% CI, 15% to 44%) of 40 patients. Neurologic function was improved in 12 (57%) of 21 patients with an abnormal assessment at baseline. The adverse event and PK profiles were consistent with previous reports for osimertinib. CONCLUSION Osimertinib showed meaningful therapeutic efficacy in the CNS and a manageable safety profile at 160 mg once daily in patients with EGFRm NSCLC and LM.

Objective\ud We conducted a feasibility trial of acupuncture in cancer patients undergoing radiotherapy treatment. The trial included training radiographers to deliver acupuncture within patients’ routine NHS care.\ud \ud Methods\ud Mixed methods pragmatic randomized parallel-group exploratory feasibility trial comparing standard care to standard care plus acupuncture.\ud \ud Results\ud Most aspects of the research design and acupuncture intervention were acceptable to the 101 participants. Participants’ valued the opportunity to receive acupuncture within their NHS care, perceived the treatment as eliciting a number of beneficial effects, and had a positive impact on their NHS cancer treatment. However, quantitative analysis of outcome measure data revealed no consistent significant differences between those receiving standard care and those receiving standard care plus acupuncture.\ud \ud Conclusion\ud It is feasible to implement acupuncture in a busy radiotherapy unit provided by specially trained radiographers. The methodology employed appears acceptable for the evaluation of acupuncture for radiotherapy patients.

TPS261 Background: For patients (pts) with HER2-overexpressing metastatic gastric cancer, trastuzumab + chemotherapy is a standard first-line option but provides only a modest overall survival (OS) benefit vs chemotherapy. T-DXd is an antibody-drug conjugate consisting of an anti-HER2 antibody, cleavable tetrapeptide-based linker, and a membrane-permeable topoisomerase I inhibitor payload. Results from a phase 1 trial showed promising antitumor activity (confirmed objective response rate [ORR], 43.2%) in pts with heavily pretreated HER2+ metastatic gastric cancer who received T-DXd (5.4 or 6.4 mg/kg; Shitara K, et al. Lancet Oncol. 2019;20:827-836). Here we describe the phase 1b/2 DESTINY-Gastric03 trial (NCT04379596) evaluating T-DXd monotherapy and combinations in pts with HER2-overexpressing gastric cancer. Methods: This is an open-label, multicenter, 2-part, phase 1b/2 study in pts with HER2-overexpressing (immunohistochemistry [IHC] 3+ or IHC 2+/in situ hybridization positive) locally advanced, unresectable or metastatic gastric or gastroesophageal junction cancer. In part 1 (dose escalation), pts who had received prior trastuzumab-containing therapy will be assigned to 1 of 5 arms: (1) T-DXd + 5-fluorouracil (5-FU); (2) T-DXd + capecitabine (C); (3) T-DXd + durvalumab; (4) T-DXd + 5-FU or C + oxaliplatin (Ox); or (5) T-DXd + 5-FU or C + durvalumab. In part 2 (dose expansion), pts with no prior treatment for metastatic disease will be randomized across 4 arms: (1) T-DXd; (2) trastuzumab + 5-FU or C + Ox or cisplatin; (3) T-DXd + 5-FU or C ± Ox; or (4) T-DXd + 5-FU or C + durvalumab. In part 2, pts will be stratified by HER2 status. Primary endpoints are safety, determination of recommended phase 2 doses (part 1), and investigator-assessed confirmed ORR per RECIST v1.1 (part 2). Secondary endpoints include confirmed ORR (part 1), disease control rate, duration of response, progression-free survival (all per investigator), OS, safety (part 2), pharmacokinetics, and immunogenicity. Clinical trial information: NCT04379596.

Metastatic spread is the single-most powerful predictor of poor outcome in Ewing sarcoma (ES). Therefore targeting pathways that drive metastasis has tremendous potential to reduce the burden of disease in ES. We previously showed that activation of the ERBB4 tyrosine kinase suppresses anoikis, or detachment-induced cell death, and induces chemoresistance in ES cell lines in vitro. We now show that ERBB4 is transcriptionally overexpressed in ES cell lines derived from chemoresistant or metastatic ES tumours. ERBB4 activates the PI3K-Akt cascade and focal adhesion kinase (FAK), and both pathways contribute to ERBB4-mediated activation of the Rac1 GTPase in vitro and in vivo. ERBB4 augments tumour invasion and metastasis in vivo, and these effects are blocked by ERBB4 knockdown. ERBB4 expression correlates significantly with reduced disease-free survival, and increased expression is observed in metastatic compared to primary patient-matched ES biopsies. Our findings identify a novel ERBB4-PI3K-Akt-FAK-Rac1 pathway associated with aggressive disease in ES. These results predict that therapeutic targeting of ERBB4, alone or in combination with cytotoxic agents, may suppress the metastatic phenotype in ES.

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

Learning Objectives After completing this course, the reader will be able to: Explain the importance of resistance to anoikis in the development of metastases.Describe the mechanisms of anoikis resistance in EFTs and osteosarcoma and their potential use in development of new therapies. CME This article is available for continuing medical education credit at CME.TheOncologist.com Detection of micrometastatic tumor cells in the bone marrow or peripheral blood of patients with Ewing family of tumors (EFTs) and osteosarcoma has been shown to correlate with poor outcome. Although one of the aims of chemotherapy is eradication of micrometastatic disease, these cells vary phenotypically from primary tumor cells and appear to be more resistant to chemotherapy. As a barrier to metastasis, cells normally undergo a form of cell death termed anoikis after they lose contact with the extracellular matrix or neighboring cells. Tumor cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while traveling through the circulation. Investigating mechanisms of resistance to anoikis, therefore, provides a valuable model to investigate regulation of micrometastatic disease. This review focuses on the current understanding of the mechanisms involved in mediating cell survival and resistance to anoikis in EFTs and osteosarcoma and discusses future studies that may help to identify novel therapeutics targeted at micrometastatic disease.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

In this study we have used the transgenic mouse model Tg2576 to analyze the involvement of anomalous loss of regulation of cdk5 and the stress kinases JNK and p38 in brain neuronal death as related to neurodegenerative disorders such as Alzheimer's disease. Previous studies on hippocampal cells led us to the discovery that the cdk5/p35 complex is activated in neurodegeneration, a finding that was confirmed later in the transgenic mouse model. Here we show a link between the cdk5 system and JNK and p38 phosphoproteins, as an alternative pathway to neuronal death. Brains of the Tg2576 transgenic mice overexpressing amyloid precursor protein exhibited immunoreactivity with the phosphoproteins p-JNK, p-p38 and the GTPase protein Rac1 surrounding neuritic plaques. A significant increase in the immunodetection of JNK and p38 phosphoproteins in the Tg2576 mouse compared with wild type controls confirmed these findings. The significant increase in co-immunoprecipitation of p-JNK, p-p38 and Rac1 proteins with cdk5 in the transgenic mouse provided evidence for these interactions. At the cellular level, p-JNK and cdk5 colocalized in the cytoplasm of the cell bodies and neurites of brain cortical areas of the transgenic mouse. The present evidence suggests a cellular link between the cdk5 system and the stress kinase JNK and p38 pathways in an in vivo model. This study sheds new light on the pathogenesis of neuronal degeneration processes such as those occurring in Alzheimer's disease.

Background: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo. Methods: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model. Results: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo. Conclusions: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols.

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

Purpose The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. Patients and Methods Forty-three stage IV and seven stage III patients were vaccinated four times with TRIMEL/DC vaccine. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and regulatory T-cell populations were determined. Overall survival and disease progression rates were analyzed using Kaplan-Meier curves and compared with historical records. Results The overall survival for stage IV patients was 15 months. More than 60% of patients showed DTH-positive reaction against the TRIMEL. Stage IV/DTH-positive patients displayed a median survival of 33 months compared with 11 months observed for DTH-negative patients (P = .0014). All stage III treated patients were DTH positive and remained alive and tumor free for a median follow-up period of 48 months (range, 33 to 64 months). DTH-positive patients showed a marked reduction in the proportion of CD4+ transforming growth factor (TGF) β+ regulatory T cells compared to DTH-negative patients (1.54% v 5.78%; P < .0001). Conclusion Our findings strongly suggest that TRIMEL-pulsed DCs provide a standardized and widely applicable source of melanoma antigens, very effective in evoking antimelanoma immune response. To our knowledge, this is the first report describing a correlation between vaccine-induced reduction of CD4+TGFβ+ regulatory T cells and in vivo antimelanoma immune response associated to improved patient survival and disease stability.

A number of psychiatric and neurodegenerative disorders, such as Alzheimer's disease, are characterized by abnormalities in the neuronal cytoskeleton. Here, we find that the enhancement in actin polymerization induced by fibrillar amyloid-beta peptide (Aβ) is associated with increased activity of Rac1/Cdc42 Rho GTPases. Rac1 upregulation involves the participation of Tiam1, a Rac guanine-nucleotide exchange factor, where Aβ exposure leads to Tiam1 activation by a Ca2+-dependent mechanism. These results point to Rho GTPases as one of the targets in Aβ-induced neurodegeneration in Alzheimer's disease pathology, with a role in mediating changes in the actin cytoskeletal dynamics.

PURPOSE. Uveal melanoma is the most common primary malignant ocular cancer in adults. This tumor has a distinct expression pattern of markers compared with cutaneous melanoma. MC1R is under study as a potential target for antitumor immunity. Because of the potential immunogenicity of MC1R, it is important to evaluate its expression on uveal melanomas.METHODS. Two novel monoclonal antibodies (MP1.1C11 and MP1.1B7) were used to examine the expression of MC1R in uveal melanomas. Tissue samples obtained from 17 patients were analyzed for expression of MC1R by immunohistochemistry. Additionally, uveal melanoma cell lines were treated with proinflammatory cytokines, after which MC1R cell surface expression was analyzed by flow cytometry.RESULTS. Results demonstrated that MC1R is expressed by uveal melanoma to a significantly greater extent than other melanoma markers. With the use of MP1.1C11 or MP1.1B7, MC1R was detected in 95% of the tested melanoma tissues, including one liver metastasis. In contrast, MART-1, S100-specific protein, and gp-100 were only expressed by 66%, 33%, and 67% of the analyzed samples, respectively. Results also demonstrated that even though MC1R is mainly located intracellularly, its cell surface expression can be promoted by cytokines such as IFN-gamma, TNF-alpha, IL-4, and IL-10.CONCLUSIONS. These observations support the inclusion of MC1R in the panel of markers for the diagnosis of uveal melanoma. Therapeutic use of MC1R-specific antibodies targeting cytokine-induced MC1R potentially requires expression of the target molecule on the surfaces of tumor cells. Data presented here support MC1R as a new marker and a putative therapeutic target for uveal melanoma.

The diagnosis of Alzheimer's disease (AD) is mainly performed by excluding other disorders with similar clinical features. In addition, an analysis of symptoms and signs, blood studies and brain imaging are major ingredients of the clinical diagnostic work-up. However, the diagnosis based on these instruments is unsatisfactory, indicating the need of a highly sensitive and reliable approaches, selective for AD and based on biological markers. Ideally, such markers should reflect the pathophysiological mechanisms of AD, which according to the current hypotheses, derive from the actions of two major protein aggregates, the extracellular beta-amyloid (Abeta) plaques and the neurofibrillary tangles. Since AD is a multifactorial disease, other factors that cause neuronal insult and that contribute to neuronal degeneration in AD include free radical and oxidative stress promoting molecules, proinflammatory cytokines and neurotoxic agents. In this context, the search for anomalous levels or changes in the molecular patterns of Abeta(1-42) or Abeta(1-40), hyperphosphorylated tau isoforms, oxidation products in the cell or cytokines such as interleukin-1 or 6 facilitates the selection of biomarkers in AD. There is clear evidence that the cerebrospinal fluid (CSF) levels of beta(1-42) are significantly reduced in AD patients as compared with senile controls, while increased levels of tau have been revealed. The CSF levels of these proteins reflect their metabolism in the central nervous system. Approaches using ELISA and immunochemical methods for the quantification of these markers in CSF have been preferentially used. Diagnosis criteria and number of patients exhibits variations in the different reports, while clinico-pathological studies are scarce. An increasing number of studies suggest that supplementary use of these CSF markers preferably in combination, adds to the accuracy of an AD diagnosis.

Most Ewing family tumor (EFT) related deaths occur as a consequence of metastatic disease. Using high-throughput Affymetrix arrays, and matched primary and metastatic tumor-derived cell line pairs, we have identified ERBB4, a member of the epidermal growth factor receptor (EGFR) family, as a novel driver of metastasis and anoikis resistance in EFTs. Gene expression profiling, mRNA analysis, and immunoblotting revealed a 2 to 4-fold increase in ERBB4 expression in metastatic compared to primary EFT cell lines. SNP profiling failed to show copy number aberrations to explain the increased ErbB4 expression in metastatic cell lines. However, epigenetic mechanisms were found to potentially regulate ERBB4 expression in the paired CHLA-9 primary and in the metastatic/chemoresistant CHLA-10 cell line derived from the same patient. Treatment with 5-aza-2α-deoxycytidine enhanced ErbB4 in CHLA-9 cells, but reduced its expression in CHLA-10, suggesting a potential role for DNA methylation in controlling ERBB4 expression in EFTs. Enhanced protein expression and ErbB4 tyrosine kinase activation were clearly identified in metastatic versus primary cells grown under anchorage independent conditions (as multicellular spheroids), which mimic micrometastases. We next evaluated the clinical relevance of these data using biopsies from EFT patients. We found a direct correlation between ErbB4 expression and reduced disease-free survival (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 431. doi:1538-7445.AM2012-431

Most Ewing sarcoma (ES)-related deaths occur as a result of metastatic disease. As a barrier to metastases, cells normally undergo a form of cell death called ‘‘anoikis” after they lose contact with the extracellular matrix or neighboring cells. Tumor cells that acquire malignant potential develop mechanisms to suppress anoikis. We previously demonstrated that ES tumors suppress anoikis and show prolonged survival as multicellular spheroids, which mimic micrometastases under anchorage-independent conditions. Here we have used the paired CHLA-9 primary tumor line established at diagnosis and the metastatic CHLA-10 line established from the same patient after four cycles of induction chemotherapy with cisplatin, doxorubicin, cyclophosphamide, and etoposide, as well as high-throughput Affymetrix whole exon array platforms to screen for potential therapeutic targets to treat metastatic disease. We have identified ErbB4, a member of the epidermal growth factor receptor (EGFR) family, as a novel mediator of anoikis resistance in ES. We detected a 4-fold increase in ERBB4, along with enhanced ErbB4 protein expression and Tyr kinase activation (confirmed by P-Tyr kinase proteome arrays) in CHLA-10 cells grown under anchorage independent conditions, compared to CHLA-9 tumor cells under the same conditions. Upregulated ErbB4 expression was further confirmed in a panel of metastatic vs. primary ES cell lines at both the mRNA and protein levels. We are currently validating these findings in the clinical setting by analyzing ErbB4 expression in paired primary vs. metastatic biopsies from ES patients. shRNA-mediated knockdown of ErbB4, or treatment with the ErbB inhibitor Lapatinib, dramatically reduced proliferation, decelerated tumor invasion, and sensitized metastatic/chemoresistant ES tumors to chemotherapeutic drugs used to treat ES patients. Taken together, our findings identify ErbB4 as a novel target for metastasis and anoikis resistance in ES. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1636. doi:10.1158/1538-7445.AM2011-1636