Regional Draining Lymph Nodes Research Articles

Regulation of respiratory CD8+ T cell immunity by suppressive monocyte-like dendritic cells

Abstract Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+ monocyte-like dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection. Supported by grant from NIH (R21 AI131093)

The Lymphatic System in Kidney Disease.

The high-capacity vessels of the lymphatic system drain extravasated fluid and macromolecules from nearly every part of the body. However, far from merely a passive conduit for fluid removal, the lymphatic system also plays a critical and active role in immune surveillance and immune response modulation through the presentation of fluid, macromolecules, and trafficking immune cells to surveillance cells in regional draining lymph nodes before their return to the systemic circulation. The potential effect of this system in numerous disease states both within and outside of the kidney is increasingly being explored for their therapeutic potential. In the kidneys, the lymphatics play a critical role in both fluid and macromolecule removal to maintain oncotic and hydrostatic pressure gradients for normal kidney function, as well as in shaping kidney immunity, and potentially in balancing physiological pathways that promote healthy organ maintenance and responses to injury. In many states of kidney disease, including AKI, the demand on the preexisting lymphatic network increases for clearance of injury-related tissue edema and inflammatory infiltrates. Lymphangiogenesis, stimulated by macrophages, injured resident cells, and other drivers in kidney tissue, is highly prevalent in settings of AKI, CKD, and transplantation. Accumulating evidence points toward lymphangiogenesis being possibly harmful in AKI and kidney allograft rejection, which would potentially position lymphatics as another target for novel therapies to improve outcomes. However, the extent to which lymphangiogenesis is protective rather than maladaptive in the kidney in various settings remains poorly understood and thus an area of active research.

English

Myiasis is the infestation of living tissues of human and animals with larvae of flies in the Order Diptera. This study determined the gross and histopathological lesions due to Cordylobia anthropophaga infestations of Dogs in Kitui County, Kenya. Four dogs identified and confirmed to have canine cutaneous myiasis (CCM) infestation were purchased for the study. All the study animals were clinically examined for skin lesions characteristic of CCM and euthanized for complete necropsy examination and histopathological sample collection. Tissue sections were collected from areas with lesions of CCM namely; skin, skeletal muscles and regional draining lymph nodes. They were preserved in 10% buffered formalin and were processed and analyzed using standard methods. Grossly, all dogs had poor body condition, patches of alopecia, emaciated and had the pathognomonic lesions for canine cutaneous myiasis (CCM) characterized by multifocal coalescent nodular lesions (harboring Cordylobia anthropophaga larvae) with 3 mm diameter central pore (furuncles) and erythematous base. Some had serous, hemorrhagic or purulent discharges from the furuncles that matted surrounding hairs. Lesions were distributed mainly on the ventral abdomen, axilla, flanks, legs, perineum and external reproductive organs. Examination of the skin revealed that the furuncular lesions extended throughout the skin thickness from epidermis to the sub-cutis and underlying pale skeletal muscles. There was regional lymphadenopathy in affected body regions. Histopathology confirmed the main lesions in all the dogs were parasitic granulomas, congested blood vessels in the surrounding tissues and eosinophilic lymphadenitis. The granulomas were located in the skin and the underlying skeletal muscles. The lesions were characterized by centrally located parasite, surrounded by connective tissue and heavy infiltration with inflammatory cells predominantly eosinophils. Draining lymph nodes had eosinophilic lymphadenitis. The reported pathological lesions resulted in unthriftiness, alopecia, lethargy and anorexia due to pain and stress resulting to emaciation and possible death. There is need to eradicate the etiological agent as it affects the wellbeing of dogs in the study area. Key words: Canine, Cordylobia anthropophaga, Myiasis, gross pathology, histopathology, Kitui County.

Fluorescence-guided lymph node sampling is feasible during up-front or delayed nephrectomy for Wilms tumor

BackgroundLymph node sampling is critical to surgical staging in Wilms tumor; failure to sample lymph nodes is associated with under-staging and an increased incidence of local relapse. However, no standard lymphatic mapping method is currently being utilized for Wilms tumor to aid identification of regional draining lymph nodes. Herein, we describe the use of fluorescence-guided lymphatic mapping for Wilms tumor. Materials and methodsTwo tertiary level referral centers independently began indocyanine green (ICG) fluorescence-guided nodal mapping. In one center, this was achieved with ipsilateral intra-parenchymal (IP) injection of ICG during minimally invasive tumor nephrectomy (MIN) following neoadjuvant chemotherapy and in the other, with Peri‑Hilar (PH) injection during upfront, open tumor nephrectomy (ON). Successful lymph node mapping was defined as the presence of fluorescence signal in draining lymph nodes. ResultsEight patients (median age of 2.5 years) underwent fluorescence-guided lymphatic mapping (four IP and four PH injection). Lymphatic mapping was successful in seven patients (88%) including each of the four patients with IP injection. ConclusionsFluorescence-guided lymphatic mapping of Wilms tumor drainage is feasible by both IP injection and PH injection techniques. However, whether lymphatic mapping improves the precision of lymph node sampling is unknown and should be studied in prospective trials.

Hypertension promotes microbial translocation and dysbiotic shifts in the fecal microbiome of nonhuman primates.

Accumulating evidence indicates a link between gut barrier dysfunction and hypertension. However, it is unclear whether hypertension causes gut barrier dysfunction or vice versa and whether the gut microbiome plays a role. To understand this relationship, we first cross-sectionally examined 153 nonhuman primates [NHPs; Chlorocebus aethiops sabaeus; mean age, 16 ± 0.4 yr; 129 (84.3%) females] for cardiometabolic risk factors and gut barrier function biomarkers. This analysis identified blood pressure and age as specific factors that independently associated with microbial translocation. We then longitudinally tracked male, age-matched spontaneously hypertensive NHPs (Macaca mulatta) to normotensives (n = 16), mean age of 5.8 ± 0.5 yr, to confirm hypertension-related gut barrier dysfunction and to explore the role of microbiome by comparing groups at baseline, 12, and 27 mo. Collectively, hypertensive animals in both studies showed evidence of gut barrier dysfunction (i.e., microbial translocation), as indicated by higher plasma levels of lipopolysaccharide-binding protein (LBP)-1, when compared with normotensive animals. Furthermore, plasma LBP-1 levels were correlated with diastolic blood pressure, independent of age and other health markers, suggesting specificity of the effect of hypertension on microbial translocation. In over 2 yr of longitudinal assessment, hypertensive animals had escalating plasma levels of LBP-1 and greater bacterial gene expression in mesenteric lymph nodes compared with normotensive animals, confirming microbes translocated across the intestinal barrier. Concomitantly, we identified distinct shifts in the gut microbial signature of hypertensive versus normotensive animals at 12 and 27 mo. These results suggest that hypertension contributes to microbial translocation in the gut and eventually unhealthy shifts in the gut microbiome, possibly contributing to poor health outcomes, providing further impetus for the management of hypertension.NEW & NOTEWORTHY Hypertension specifically had detrimental effects on microbial translocation when age and metabolic syndrome criteria were evaluated as drivers of cardiovascular disease in a relevant nonhuman primate model. Intestinal barrier function exponentially decayed over time with chronic hypertension, and microbial translocation was confirmed by detection of more microbial genes in regional draining lymph nodes. Chronic hypertension resulted in fecal microbial dysbiosis and elevations of the biomarker NT-proBNP. This study provides insights on the barrier dysfunction, dysbiosis, and hypertension in controlled studies of nonhuman primates. Our study includes a longitudinal component comparing naturally occurring hypertensive to normotensive primates to confirm microbial translocation and dysbiotic microbiome development. Hypertension is an underappreciated driver of subclinical endotoxemia that can drive chronic inflammatory diseases.

Lymph node mapping in gastric cancer: A pilot study in Western patients.

192 Background: Adequate surgical lymphadenectomy is integral to the staging and treatment of gastric cancer. A number of Asian studies have explored the utility of lymph node (LN) mapping in gastric cancer but this in not commonly performed in Western countries. We sought to evaluate the utility and safety of LN mapping in Western patients. Methods: We conducted a pilot study of 13 patients with histologically proven non-metastatic gastric adenocarcinoma that received endoscopic peri-tumoral indocyanine green fluorescence (ICG) injections immediately prior to surgical resection to facilitate LN mapping. Illumination with ICG around the primary tumor, in lymphatic basins, tracts, and lymph nodes were confirmed by the PINPOINT system on recorded videos. Descriptive statistical analysis was performed. Results: Among the 13 patients enrolled, median age was 75 years and 7 were men. On pathologic review, 15% were T1, 54% were T2, and 69% had were node negative. Tumours were located in the proximal third in 1 patient, middle third in 2 patients and distal third in 10 patients. All patients had a laparoscopic subtotal gastrectomy and 10 patients had a D2 lymphadenectomy. 5 patients received preoperative and 7 received postoperative chemotherapy. The median number of LNs harvested was 26 [IQR 24-34]. Video confirmation of ICG mapping of the primary tumor, lymphatic basins, tracts, and LNs were obtained in all patients. All LNs identified with ICG uptake were removed with surgical lymphadenectomy. ICG mapped LNs fell outside the D1 distribution in 100% and outside the D1-plus distribution in 54% of cases. ICG mapped LNs were within the D2 distribution in all cases. No ICG related allergic reactions or procedural complications were observed. Postoperative complications included 2 grade A pancreatic fistulas, 1 gastrointestinal bleed, and 1 NSTEMI. Peri-operative morality was zero. Conclusions: We demonstrate ICG lymph node mapping as a safe and useful technique for identifying regional draining lymph nodes and for primary tumor localization in Western patients. The majority of cases found LNs draining outside the D1 and D1+ distributions, highlighting the importance of appropriate lymphadenectomies in gastric cancer.

Regulation of Respiratory CD8+ T cell Immunity by Suppressive Monocyte Derived Dendritic Cells (moDCs)

Abstract Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+monocyte-derived dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection. Ongoing experiments seek to uncouple moDC interactions with CD8+ T cells in specific sites in order to better understand, and potentially beneficially modulate, CD8+ T cell immunity within the lung.

Unconventional immunotherapy with an unconventional target

Pritumumab, a natural human IgG1 kappa antibody was obtained from a regional draining lymph node of a patient with cervical carcinoma through traditional hybridoma technology. Specificity analysis of the target antigen, an altered form of vimentin called, ecto-domain vimentin (EDV), shows it to be limited to cell surface expression on cancer cells. Clinically, 249 brain cancer patients were treated with a low dose pritumumab regimen, either at 1 mg once a week or 1 mg twice a week, and of those evaluated overall response rates of between 25-30% were seen with several complete and partial responses. A clinical trial assessing higher doses of pritumumab as a therapeutic for brain cancer is expected to begin this year. Overall, these data together suggest pritumumab is suitable for further development as an anti-tumor therapeutic.

Effect and Safety of Radiation Therapy Boost to Extramesorectal Lymph Nodes in Rectal Cancer

PurposeIn rectal cancer, the presence of extramesorectal/lateral pelvic lymph node (LPN) is associated with higher risk of locoregional and distant recurrences. LPNs are not typically resected during a standard total mesorectal excision (TME) procedure, and the optimal management for these patients is controversial. We assessed the safety and efficacy of adding a radiation therapy boost to clinically positive LPN during neoadjuvant chemoradiation therapy for rectal cancer. Methods and MaterialsWe analyzed nonmetastatic, lymph node positive rectal adenocarcinoma patients treated with neoadjuvant chemoradiation therapy followed by TME between May 2011 and February 2018. Patients without LPN involvement received external beam radiation therapy (45 Gy in 25 fractions) to the primary tumor and regional draining lymph node basins followed by a boost (5.4 Gy in 3 fractions) to gross disease. Patients with clinically positive LPN that would not be removed during TME received an additional boost (up to a total dose between 54.0 and 59.4 Gy) to the involved LPNs. We compared locoregional control, overall survival, progression-free survival, and treatment-related toxicity between these 2 groups. ResultsFifty-three patients were included in this analysis with median follow-up of 30.6 months for the LPN– group (n = 41) and 19.9 months for the LPN+ group (n = 12). There was no difference in 3-year overall survival (90.04% vs 83.33%, P = .890) and progression-free survival (80.12% vs 80.21%, P = .529) between the 2 groups. We did not observe any LPN recurrences. There were no differences in rates of acute grade 3+ or chronic toxicities. ConclusionsDespite the well-documented negative prognostic effect of LPN metastasis, we observed promising outcomes for LPN+ patients treated with an additional radiation boost. Our results suggest that radiation therapy boost to clinically involved, unresected LPN is an effective treatment approach with limited toxicity. Additional studies are needed to optimize treatment strategies for this unique patient subset.

The natural human IgG1 mAb Pritumumab targets cell surface expressed vimentin and inhibits tumor growth

Abstract Cancer patients generate tumor-specific B lymphocytes which can be isolated to develop human mAbs against tumor-associated antigens. Pritumumab (also referred to as CLNH-11, CLN-IgG, or ACA-11) is a classic example of a natural human anti-cancer antibody. It is a natural human IgG1 kappa antibody developed by human hybridoma technology, using B lymphocytes isolated from a regional draining lymph node of a patient with cervical carcinoma. Phase II clinical trials with pritumumab have demonstrated an objective response as a therapeutic antibody for glioblastoma. Here we demonstrate pritumumab binds cell surface expressed vimentin, also referred to as ecto-domain vimentin (EDV). Vimentin is an intracellular cytoskeletal protein overexpressed during epithelial-to-mesenchymal transition (EMT), a process integral to cancer cell metastasis. The potential of pritumumab as a therapeutic antibody targeting glioblastoma was evaluated. We demonstrate pritumumab binding to patient glioblastoma cells and antibody binding to these cells induces antibody-dependent cell-mediated cytoxicity (ADCC). Furthermore, pritumumab effectively inhibited glioma tumor growth in a xenograft mouse model. Overall, these data provide pre-clinical validation of pritumumab mAb as a therapeutic for glioblastoma.

Regional Draining Lymph Nodes: Considerations for Medical Device Studies.

Lymph nodes and associated lymphatics filter extracellular fluid and lymph to maintain tissue-fluid balance and detect distant tissue injury. Examination of regional draining lymph nodes (RDLs; lymph nodes that drain the route of article dosing) is an important step in detecting immunotoxicity and other associated changes during general toxicology studies. Similarly, evaluation of RDLs is often a key component of evaluating medical devices. Nonclinical medical device studies can present challenges for RDL evaluation, due to the wide variety of tissues and organs that are implanted with devices, the potential for wear debris/degradation products, and the likely disruption of normal lymphatic drainage by surgical procedures. This article discusses concepts for consideration when designing a nonclinical medical device study that includes the macroscopic evaluation, collection, histologic processing, microscopic assessment, and documentation of findings within RDLs. References describing RDLs for common implantation sites are reported, as are considerations for specific tissues and species commonly used in medical device biocompatibility and functional testing.

Pritumumab, the first therapeutic antibody for glioma patients.

Immunotherapy is now at the forefront of cancer therapeutic development. Gliomas are a particularly aggressive form of brain cancer for which immunotherapy may hold promise. Pritumumab (also known in the literature as CLNH11, CLN-IgG, and ACA-11) was the first monoclonal antibody tested in cancer patients. Pritumumab is a natural human monoclonal antibody developed from a B lymphocyte isolated from a regional draining lymph node of a patient with cervical carcinoma. The antibody binds ecto-domain vimentin on the surface of cancer cells. Pritumumab was originally tested in clinical trials with brain cancer patients in Japan where it demonstrated therapeutic benefit. It was reported to be a safe and effective therapy for brain cancer patients at doses 5-10 fold less than currently approved antibodies. Phase I dose escalation clinical trials are now being planned with pritumumab for the near future. Here we review data on the development and characterization of pritumumab, and review clinical trails data assessing immunotherapeutic effects of pritumumab for glioma patients.

An in Vitro and in Vivo Study of the Effect of Dexamethasone on Immunoinhibitory Function of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) represent a promising cell source for patient-specific cell therapy. We previously demonstrated that they display an immunomodulatory effect on allergic airway inflammation. Glucocorticoids are powerful anti-inflammatory compounds and widely used in the therapy of allergic diseases. However, the effect of glucocorticoids on the immunomodulatory function of iPSC-MSCs remains unknown. This study aimed to determine the effect of dexamethasone (Dex) on the immunomodulatory function of iPSC-MSCs in vitro and in vivo. A total of three human iPSC-MSC clones were generated from amniocyte-derived iPSCs. Anti-CD3/CD28-induced peripheral blood mononuclear cell (PBMC) proliferation was used to assess the effect of Dex on the immunoinhibitory function of iPSC-MSCs in vitro. Mouse models of contact hypersensitivity (CHS) and allergic airway inflammation were induced, and the levels of inflammation in mice were analyzed with the treatments of iPSC-MSCs and Dex, alone and combined. The results showed that Dex did not interfere with the immunoinhibitory effect of iPSC-MSCs on PBMC proliferation. In CHS mice, simultaneous treatment with Dex did not affect the effect of iPSC-MSCs on the inflammation, both in regional draining lymph nodes and in inflamed ear tissue. In addition, co-administration of iPSC-MSCs with Dex decreased the local expression of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the ears of CHS mice. In the mouse model of allergic airway inflammation, iPSC-MSC treatment combined with Dex resulted in a similar extent of reduction in pulmonary inflammation as iPSC-MSCs or Dex treatment alone. In conclusion, Dex does not significantly affect the immunomodulatory function of iPSC-MSCs both in vitro and in vivo. These findings may have implications when iPSC-MSCs and glucocorticoids are co-administered.

Abstract 3828: Pritumumab mAb binds cell surface expressed vimentin on pancreatic cancer cells and inhibits tumor growth

Abstract Monoclonal antibodies (mAbs) as therapeutics for cancer have shown some success in the clinic. These biologics are useful if targeting a cancer specific epitope. Cancer patients can generate tumor-specific B lymphocytes which can be isolated to develop human mAbs against tumor-associated antigens. The best source of anti-tumor antibodies is from sentinel lymph nodes. Pritumumab (also referred to as CLNH-11, CLN-IgG, or ACA-11) is a classic example of a natural human anti-cancer antibody. It is a natural human IgG1 kappa antibody developed by the human hybridoma technology, using B lymphocytes isolated from a regional draining lymph node of a patient with cervical carcinoma. In the original patient from whom the B lymphocyte was isolated, the bioavailability of the target antigen induced a natural immune response resulting in the generation of the pritumumab IgG. The target antigen recognized by pritumumab is cell surface expressed vimentin, also referred to as ecto-domain vimentin (EDV). Vimentin is an intracellular cytoskeletal protein overexpressed during epithelial-to-mesenchymal transition (EMT), a process integral to cancer cell metastasis. Pancreatic cancer is a deadly disease with poor prognosis. No more than 6% of pancreatic cancer patients will survive beyond five years after diagnosis. There is a clear unmet need to help improve outcomes for patients. (A) Purpose of the study: to examine if pritumumab mAb can target pancreatic cancer cells and inhibit pancreatic tumor growth. (B) Experimental Procedures: Immunohistochemical analysis with horseradish peroxidase (HRP)-conjugated pritumumab on pancreatic cancer patient tissue sections along with immunofluorescence, flow cytometry, and In-Cell ELISA to demonstrate antibody binding. Antibody-dependent cell-mediated cytoxicity (ADCC) was performed to determine mechanism of action. In vivo subcutaneous xenograft mouse model treated with pritumumab to assess efficacy of the antibody for tumor inhibition. (C) Results: In this study we demonstrate pritumumab binds to the cell surface of patient pancreatic cancer tissue and binds the surface of established pancreatic cancer cells in culture. We show antibody binding to these cells induces antibody-dependent cell-mediated cytoxicity (ADCC). Furthermore, pritumumab effectively inhibits pancreatic tumor growth in a xenograft mouse model. (D) Statement of Conclusions: overall, these data provide pre-clinical validation of pritumumab mAb as a therapeutic for pancreatic cancer. Citation Format: Ivan Babic, Natsuko Nomura, Eric Glassy, Elmar Nurmemmedov, Venkata Yenugonda, Mark Glassy, Santosh Kesari. Pritumumab mAb binds cell surface expressed vimentin on pancreatic cancer cells and inhibits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3828.

Merkel cell carcinoma of the eyelid and orbit

The incidence of Merkel cell carcinoma has tended to increase worldwide in recent years. Merkel cell carcinoma is a rare tumor of the skin that occurs mainly in the sun exposed sites. The malignant reversal of Merkel cells is currently associated with an infection caused by a Merkel cell polyomavirals. In some cases, the disease may have a relatively inconspicuous clinical picture in the initial phase, which is in contrast to its extensive microscopic propagation. For this reason, the risk of late diagnosis or insufficient primary surgery is increased. The diagnostic standard is histological and, in particular, immunohistochemical examination of tumor tissue samples. Merkel cell carcinoma is a marked tendency to local recurrence and early development of metastases in regional lymph nodes, followed by generalization. The basis of treatment is radical excision of the tumor by in most cases by adjuvant radiotherapy targeted at primary place of occurrence and the area of regional draining lymph nodes. The effectiveness of different chemotherapeutic protocols in Merkel cell carcinoma is mostly low and the median survival is low. From a prognostic point of view, Merkel cell carcinoma plays the most important role of staging the tumor at the time of capture. The suspected lesions in the area around the eye, eyelid and orbit need to indicate adequate therapeutic approach that the detection of the disease at the earliest stage. The authors describe the clinical experience in 2 patients with Merkel cell carcinoma of the eyelid and orbit. Key words: eyelid tumors, tumors of the orbit, Merkel cell carcinoma.

Phase 2 Study of Adjuvant Chemoradiation Therapy Using Docetaxel/Cisplatin/5-Fluorouracil Before and After Intensity-Modulated Radiation Therapy With Concurrent Docetaxel in Patients With Completely (R0) Resected Gastric Carcinoma

Complete surgical R0 resection has traditionally been considered the only curative treatment in localized gastric cancer (GC). The INT 0116 study has demonstrated a significant survival benefit for completely resected (R0) GC patients treated with a fluorouracil/leucovorin chemo-radiotherapy regimen. However, this regimen is also toxic and less effective in terms of distant disease control. Therefore, a more efficacious and safer regimen is urgently needed. The eligibility criteria included histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction, complete resection of the tumor defined as surgical resection performed with curative intent and resulting in negative resection margins, disease stage IB through IV (M0). Patients received up to two 21-day cycles of post-operative adjuvant pre- and post-radiation DCF chemotherapy (docetaxel 37.5 mg/m2 on days 1 and 8, cisplatin 25 mg/m2 on days 1-3, and a continuous infusion of fluorouracil 750 mg/m2 on days 1-5), respectively. Chemo-radiotherapy between pre- and post-radiation chemotherapy was initiated on day 43 and consisted of intensity-modulated radiotherapy (45 Gy) plus concurrent docetaxel 20 mg/m2 weekly for 5 weeks. The clinical tumor volume (CTV) included the tumor bed, anastomoses, and stumps, regional draining lymph nodes. The planning target volume (PTV) consisted of the CTV plus a 0.5-cm margin in all directions to account for daily patient set-up variation. The primary end point was PFS. This single-stage phase II trial was designed to have 93% power to detect a 20% improvement in the 20-month PFS rate from 50 to 70% with a two-sided significance level of exactly 0.05. The accrual goal was 55 patients. A total of 55 patients were evaluated and 76% (42) of patients completed the prescribed therapy. The median age was 59 years (range, 28–75 years). The majority of patients had locally advanced disease (stage T3 or T4, 71%; N1–3, 93%). With a median follow-up of 61 months, the 20-month PFS rate was 75% (95% confidence interval [CI] = 64–86%). The 3- and 5- year progression-free survival rates were 67% (95% CI: 54%-80%) and 59% (95% CI: 46%-72%), respectively; and the 3- and 5-year overall survival rates were 72% (95% CI: 60%-84%) and 61% (95% CI: 48%-74%), respectively. Of 21 documented sites of first treatment failure, local-regional relapse occurred in 5 patients, distant relapse including peritoneal carcinomatosis was observed in 13 patients, and 3 patients had both local-regional and distant recurrences. The most common grade 3 or greater toxicity, during the chemotherapy phase, was neutropenia (24%). Common grade 3/4 toxicities during concurrent chemo-radiotherapy were nausea (32%), vomiting (26%), fatigue (15%), and anorexia (19%). These results demonstrate that this adjuvant regimen is active with an acceptable toxicity profile. A randomized phase 3 trial comparing the INT-0116 chemo-radiotherapy regimen with this regimen is underway.

Abstract 5611: Use of ecto-domain vimentin to target brain cancers

Abstract Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. For brain cancers, immunohistological analysis showed the binding of pritumumab to EDV to be restricted to tumor cells and not normal cells and tissues suggesting EDV may be a useful biomarker. A recombinant version of the mAb was made using the GPEx® system in CHO cells that showed comparable binding activity by flow cytometry, immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC; EC50 of 39.6ng/ml) activity. Further studies include establishing xenograft models with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth of both brain cancer stem cells as well as pancreatic cancer in nude mice but not in SCID mice suggesting an active immune response is necessary for optimal treatment. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest that EDV is a suitable target and biomarker for brain cancers. Citation Format: Ivan Babic, Rajesh Mukthavaram, Pengfei Jiang, Eric Glassy, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Use of ecto-domain vimentin to target brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2017-5611

Pritumumab binding to glioma cells induces ADCC and inhibits tumor growth.

e14004 Background: Pritumumab is a natural human IgG1 kappa antibody originally isolated from a regional draining lymph node of a patient with cervical carcinoma. This antibody binds ectodomain vimentin on the surface of tumor cells and has demonstrated some benefit to glioblastoma patients in limited clinical trials. We wanted to determine if pritumumab inhibits glioma growth in vivo and if binding to glioma cells induces cell-mediated immunity. Methods: Pritumumab was used in flow cytometry experiments with several glioma cell lines and patient-derived neurosphere lines. Antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay was used with glioma target cells. Xenograft studies were performed in mice with and without intact B- and NK- cells. Results: We performed flow cytometry using pritumumab antibody and demonstrate binding of pritumumab to the surface of glioma cells and patient-derived glioma initiating cells. We observed significant induction of ADCC by pritumumab binding to glioma cells. Xenograft studies demonstrated pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Intact cell-mediated immunity was necessary for pritumumab’s anti-tumor effect. Analysis of a blood brain barrier model showed significant binding of pritumumab in brain tumor areas and minimal distribution in normal brain tissues suggesting the antibody can cross the blood brain barrier. Conclusions: Our data demonstratepritumumab binds glioma cells in vitro and can induce ADCC. In addition, pritumumab can limit the growth of xenograft glioma tumors in vivo only in the presence of intact cell-mediated immunity. Together these data suggest pritumumab is suitable for development as an anti-tumor therapeutic.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells.

Within the ruminant system, several possibilities exist to generate dendritic cells migrating out from the tissue into the regional draining lymph nodes as afferent lymph dendritic cells (ALDCs). Here, we analyzed toll-like receptor (TLR) 1–10 mRNA expression by using quantitative real-time PCR in highly purified subsets of bovine ALDC. As TLR expression may be influenced by pathogens or vaccines and their adjuvant, it is necessary to understand what TLRs are expressed in a steady-state system to elucidate specific differences and to potentially optimize targeted vaccines. In this study, we have assessed the TLR expression profiles of the four main bovine ALDC subsets [cDC1 and cDC2 (subsets 2–4)]. We demonstrate differences in TLR expression between the four subsets that may reflect the ability of these cells to respond to different pathogens or to respond to adjuvants.

Analysis of hybridoma and CHO-derived pritumumab for therapeutic applications.

71 Background: Pritumumab, a natural huma IgG1 kappa antibody was obtained from a regional draining lymph node of a patient with cervical carcinoma through traditional hybridoma technology. Methods: Both cell lines and tissues were processed and analyzed for FACS analysis, Western blots, and immunohistochemistry. Results: Specificity analysis of the target antigen, an altered form of vimentin (ecto-domain vimentin (EDV)) shows it to be limited to cell surface expression on cancer cells. Clinically, 249 Japanese brain cancer patients were treated with a low dose pritumumab regimen, either at 1mg once a week or 1mg twice a week, and overall response rates of between 25-30% were seen with several complete and partial responses. A recombinant version of the mAb was made using the GPEx system in CHO cells. In a series of comparable studies the CHO mAb was compared with the original hybridoma. Binding specificity with both flow cytometry and immunohistochemical (IHC) analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activity confirms the comparability of the two versions. IHC analysis suggests restricted binding to tumor cells and not normal cells and tissues. Further studies include establishing a xenograft model with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth in nude mice but not in SCID mice. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Conclusions: Overall, these data together suggest pritumumab is suitable for development as an anti-tumor therapeutic.