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Background: Cytokines are being explored as a therapeutic strategy to modulate the tumor microenvironment and facilitate immunotherapy benefit in breast cancer. Here, we investigate a locoregional therapeutic approach whereby cytokines (IRX-2) are administered into the subcutaneous peri-areolar tissue (in an anatomic distribution similar to sentinel lymph node mapping) to facilitate immune cell recruitment/activation within the draining lymph nodes and tumor in ESBC. IRX-2 is derived from ex vivo phytohemagglutinin-stimulated lymphocytes and contains multiple cytokines including IL-1β, IL-2, TNF-α, IFN-γ, IL-6, IL-8, and GM-CSF, with stable concentrations from lot to lot. Preclinically, IRX-2 activates T-cells and natural killer (NK) cells, facilitates antigen presentation, and enhances activity of anti-PD-1/L1 in a SCC7 model. In a preceding head/neck squamous cell carcinoma phase I trial, perilymphatic IRX-2 was safe and increased TILs. Here, we report the final clinical results of a phase Ib trial evaluating the feasibility and immunologic activity of IRX-2 in ESBC. Methods: Beginning 21 days prior to surgical resection, enrolled operable patients with stage I-III ESBC (all subtypes) received the pre-operative IRX-2 regimen consisting of a single low-dose cyclophosphamide (300 mg/m2 to facilitate T-regulatory cell depletion), followed by 10 days of subcutaneous peri-areolar IRX-2 injections into the affected breast (1 mL × 2 at tumor axis and at 90°). Endpoints were feasibility (primary endpoint), stromal TIL (sTIL) count (pre-treatment versus post-treatment, blinded average of two pathologist reads using San Antonio H&E sTIL guidelines), PD-L1 expression (Nanostring) and enumeration of peripheral immune cells by flow cytometry. Results: All patients (n=16/16) completed and tolerated the regimen with no surgical delays or treatment-attributed grade III/IV toxicities. Common adverse events (occurring in >15% subjects) attributed to IRX-2 injections were: injection site reaction (grade 1, n=8/16), bruising (grade 1, n=7/16), and pain (grade 1, n=3/16). Common adverse events attributed to low-dose cyclophosphamide were: fatigue (grade 1, n=5/16) and nausea (grade 1/2, n=3/16). Treatment was associated with an increase in sTIL score (Wilcoxon signed-rank p=.04), with 4/10 sTIL-low tumors (0-10% score) re-categorized to sTIL-moderate (11-50% score). Increases in PD-L1 RNA expression were observed (Wilcoxon signed-rank p=.04) in 12/16 tumors (median 57% increase, range: -53% to 185% increase), as well as increases in Nanostring NK and Th1 cell signatures. In blood, increases in CD4 and CD8 effector T-cell activation (ICOS, HLA-DR, and CD38) and T-reg depletion were observed. Conclusions: IRX-2 was well tolerated with preliminary evidence of sTIL increase, PD-L1 upregulation, and peripheral lymphocyte activation. Based upon these data and preclinical evaluations demonstrating synergy with checkpoint inhibition, the IRX-2 regimen is being evaluated for clinical efficacy in conjunction with pembrolizumab and neoadjuvant chemotherapy (doxorubicin, cyclophosphamide, paclitaxel) in patients with stage II-III triple negative breast cancer. Citation Format: Pucilowska J, Egan JE, Berinstein NL, Moxon N, Aliabadi-Wahle S, Imatani JH, Conlin A, Acheson A, Massimino K, Martel M, Campbell M, Wu Y, Sun Z, Redmond W, Shah M, Urba WJ, Page DB. Perilymphatic IRX-2 cytokine therapy to enhance tumor infiltrating lymphocytes (TILs) and PD-L1 expression preceding curative-intent therapy in early stage breast cancer (ESBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-12.

Background Treatment of recurrent/refractory (r/r) DLBCL remains an unmet clinical need, and new effective and well-tolerated treatments are required. DPX-Survivac is a unique T cell activation therapy that targets survivin-expressing tumor cells and has shown anti-tumor activity in clinical trials. This trial is evaluating a novel immunotherapy combination with DPX-Survivac, intermittent low dose CPA and pembrolizumab. Methods ‘SPiReL’ is a Phase 2 non-randomized, open label, efficacy and safety study of a novel immunotherapy combination with DPX-Survivac (a unique T cell activation therapy that targets survivin-expressing tumor cells), intermittent low dose CPA and pembrolizumab, treatment regimen as described in figure 1. Subjects with r/r incurable DLBCL and survivin expression are eligible for participation. This study was approved by the Ontario Cancer Research Ethics Board, approval number 0981.ORR is assessed by modified Cheson criteria. For translational analyses, baseline and on-treatment PBMCs, along with tumor biopsy samples are collected from each subject. Survivin-specific systemic T cell responses are assessed using IFNγ-ELISPOT assay and tumour immune-infiltrate profile by multiplex-IHC. Results Twenty-two subjects have been enrolled to date, 19 are included in the intent to treat (ITT) population and 11 subjects are evaluable in the per protocol (PP) population. In the PP, the ORR is 63.6% including 3 CRs (27.3%), 4 PRs (36.4%) and the DCR is 81.8% (9/11). In the ITT, the ORR is 35% (7/19), and DCR is 52.0% (10/19). Preliminary results show that non-GCB subjects had a higher proportion of clinical response (4/8, 50%), compared to 3/10 (30%) in GCB subjects.DPX-Survivac-induced T cell responses were observed in 8/19 subjects (42.1%) including 6 subjects with clinical response (PR, CR), one SD and one PD. Multiplex-IHC analyses demonstrated baseline tumor PD-L1 expression in 6/7 subjects with a clinical response (85.7%, p Conclusions DPX-Survivac, intermittent low-dose CPA and pembrolizumab is generally well tolerated and can induce clinical responses in subjects with r/r DLBCL (7/11, 63.6% of evaluable subjects), including subjects with both non-GCB and GCB subtypes. Pre-treatment biopsies of clinical responders were characterized by higher baseline tumor PD-L1 expression and CD4 and CD8 infiltration. Extending this exploratory data in a larger cohort may define a r/r DLBCL patient population with a higher likelihood to respond to this novel combination immunotherapy. Trial Registration NCT03349450 Ethics Approval This study was approved by the Ontario Cancer Research Ethics Board, approval number 0981.

Elevated levels of the carcinoembryonic antigen (CEA; CEACAM5) in the serum of colorectal cancer (CRC) patients represent a clinical biomarker that correlates with disease recurrence. However, a mechanistic role for soluble CEA (sCEA) in tumor progression and metastasis remains to be established. In our study, we report that sCEA acts as a paracrine factor, activating human fibroblasts by signaling through both the STAT3 and AKT1-mTORC1 pathways, promoting their transition to a cancer-associated fibroblast (CaF) phenotype. sCEA-activated fibroblasts express and secrete higher levels of fibronectin, including cellular EDA+ -fibronectin (Fn-EDA) that selectively promote the implantation and adherence of CEA-expressing cancer cells. Immunohistochemical analyses of liver tissues derived from CRC patients with elevated levels of sCEA reveal that the expression of cellular Fn-EDA co-registers with CEA-expressing liver metastases. Taken together, these findings indicate a direct role for sCEA as a human fibroblast activation factor, in priming target tissues for the engraftment of CEA-expressing cancer cells, through the differentiation of tissue-resident fibroblasts, resulting in a local change in composition of the extracellular matrix.

Head and neck squamous cell carcinoma (HNSCC) is an immunosuppressive malignancy characterized by tumor-driven immune-system abnormalities that contribute to disease progression. For patients with surgically resectable HNSCC, treatment is often curative surgery followed by irradiation or chemoradiation in high-risk settings to reduce the risk of recurrence. Poor survival and considerable morbidity of current treatments suggest the need for new therapeutic modalities that can improve outcomes. Defects in antitumor immunity of HNSCC patients include suppressed dendritic cell (DC) maturation, deficient antigen-presenting cell function, compromised natural killer (NK)-cell cytotoxicity, increased apoptosis of activated T lymphocytes, and impaired immune-cell migration to tumor sites. Strategies for relieving immunosuppression and restoring antitumor immune functions could benefit HNSCC patients. IRX-2 is a primary cell-derived biologic consisting of physiologic levels of T-helper type 1 cytokines produced by stimulating peripheral blood mononuclear cells of normal donors with phytohemagglutinin. The primary active components in IRX-2 are IL2, IL1β, IFNγ, and TNFα. In vitro, IRX-2 acts on multiple immune-system cell types, including DCs, T cells, and NK cells, to overcome tumor-mediated immunosuppression. In clinical settings, IRX-2 is administered as part of a 21-day neoadjuvant regimen, which includes additional pharmacologic agents (low-dose cyclophosphamide, indomethacin, and zinc) to promote anticancer immunoresponses. In a Phase IIA trial in 27 patients with surgically resectable, previously untreated HNSCC, neoadjuvant IRX-2 increased infiltration of T cells, B cells, and DCs into tumors and was associated with radiological reductions in tumor size. Event-free survival was 64% at 2 years, and overall 5-year survival was 65%. Follow-up and data analysis are under way in the multicenter, randomized, Phase IIB INSPIRE trial evaluating the IRX-2 regimen as a stand-alone therapy for activating the immune system to recognize and attack tumors.

Five-year overall survival for high-risk Follicular Lymphoma International Prognostic Index follicular lymphoma is only approximately 50% compared with 90% for low risk. To evaluate an approach to improve upon this poor outcome, we completed an exploratory phase II trial of intensified treatment for patients with intermediate and high-risk follicular lymphoma. Front-line treatment with chemo-immunotherapy consisting of rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone was followed by radio- immunotherapy with 90-Yttrium ibritumomab tiuxetan consolidation, and 2 years of rituximab maintenance. The 5-year overall survival for intermediate and high-risk patients was 88% and 83%, respectively. Of 33 enrolled patients, 3 were off study before receiving radio-immunotherapy. Three months post radio-immunotherapy, 28/33 (85%) patients had achieved complete response including 6 patients who had only a partial response to chemo-immunotherapy and converted to complete response after radio-immunotherapy. The 5-year progression-free survival for intermediate and high risk was 79% and 58%, respectively. Nine of 19 patients with molecular markers patients remain in molecular and clinical complete remission with a median follow-up of 48 months (range 3-84 months). Post radio-immunotherapy, hematologic toxicities were mostly grade 1 and 2. However, asymptomatic grade 3 or 4 thrombocytopenia and neutropenia occurred in 11%-36% and 10%-24% of patients, respectively. Myelodysplastic syndrome occurred in 1 patient 4 years post treatment. Whereas many patients had prolonged B-cell reduction and low immunoglobulin levels post treatment, previous immunities to rubella were maintained. More aggressive upfront approaches such as this may benefit higher risk follicular lymphoma, but confirmatory trials are required. http://www.clinicaltrials.gov: NCT01446562.

Background: Recurrent/refractory (r/r) DLBCL presents a major treatment challenge, especially in the setting of patients who are ineligible for, or relapsing after, potentially curative treatments such as autologous stem cell transplant (ASCT) and chimeric antigen receptor T cell (CAR-T) therapy. Efficacious treatment options that are well-tolerated and easily accessible in this population represent a critical unmet medical need. DPX-Survivac is a targeted T cell activation therapy against cancers expressing survivin. Survivin plays an essential role in cancer biology and represents a target of choice to disrupt tumour progression. DPX-Survivac's mechanism of action relies on its ability to generate robust and durable survivin-specific T cells that migrate to, infiltrate and kill tumour cells. DPX-Survivac is administered with intermittent, low dose cyclophosphamide (CPA) used as an immunomodulator. In nonclinical studies, treatment with DPX-Survivac increases PD-L1 and PD-1 expression providing the rationale for combination with pembrolizumab. Methods: "SPiReL" is a Phase 2 non-randomized, open label, efficacy and safety study. Subjects with r/r DLBCL with confirmed survivin expression are eligible for participation. Subjects must also be ineligible for potentially curative therapy. The treatment and testing regimen is shown in the figre below. The primary objective of SPiReL is to document a minimum Objective Response Rate (ORR) of 24% (in 6/25 subjects) using the modified Cheson criteria (2007). Secondary objectives include safety, duration of response and time to next treatment. Exploratory endpoints include T cell response, tumour immune cell infiltration, and biomarker analysis. Results: At data cut-off, 22 subjects have been enrolled in the study. The median age is 75.5 years (50-82). Thirteen of 22 subjects (59.1%) are GCB sub-type, 8 subjects (36.4%) are non-GCB and 1 subject (4.5%) has primary cutaneous DLBCL, leg type. The median number of prior therapies is 2 (1-7), with 4 subjects having previously undergone ASCT and 5 subjects with transformed disease. Of the 22 enrolled subjects, 8 are not evaluable per protocol for clinical efficacy due to early disease progression. Four subjects are active on treatment, 3 of which have not yet reached the first time point for assessment. Clinical outcome was analyzed for the Full Analysis Set (FAS) (n=19) and in the Per Protocol (PP) analysis (n=11). Of 11 subjects in the PP, 7 subjects (63.6%) have achieved an objective response, meeting the study's primary endpoint; 3 subjects (27.3%) with a CR and 4 subjects (36.4%) with a PR. Three subjects (27.3%) achieved SD and thus clinical benefit was demonstrated in 10/11 (90.9%) of evaluable subjects. Two subjects (18.2%) have completed the 1 year treatment period, 8 subjects (72.7%) discontinued treatment due to disease progression, and 1 subject (9.2%) discontinued treatment due to an unrelated Adverse Event (AE). Including the entire FAS, the objective response rate was 7/19 (36.8%). This treatment combination is well-tolerated, only 11% of Treatment Related AEs (TRAEs) were assessed as Grade 3 or higher. The majority of the Grade 1 and 2 TRAEs were Injection Site Reactions (ISRs) related to DPX-Survivac. Ten serious AEs were reported, of which 3 were considered related to study treatment. No subjects have discontinued study treatment due to a TRAE. Analyses of peripheral blood T cell responses to survivin by ELISpot assay shows that 7/7 subjects who achieved an objective response have survivin-specific T cell response. One subject with PD also showed a survivin-specific T cell response, supporting the mechanism of action and the role of DPX-Survivac in anti-tumor activity. Summary: DPX-Survivac and low dose CPA in combination with pembrolizumab, demonstrates promising clinical activity in recurrent/refractory DLBCL with 10/11 (90.9%) of evaluable subjects deriving clinical benefit with minimal toxicity. The primary endpoint of this study has been reached with 7/11 (63.6%) of evaluable subjects achieving an objective response warranting further exploration of DPX-Survivac in this population. Enrollment is continuing to further define the patient population most likely to benefit from this well-tolerated therapy. Figure 1 Disclosures Bence-Bruckler: Merck: Membership on an entity's Board of Directors or advisory committees. Forward:Seattle Genetics: Research Funding; IMV: Research Funding; Merck: Research Funding; Astellas: Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; IMV: Membership on an entity's Board of Directors or advisory committees; Calgene: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Stewart:Roche: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Sandoz: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria; Teva: Honoraria. Bramhecha:IMV Inc.: Current Employment. Conlon:IMV Inc.: Current Employment. OffLabel Disclosure: Keytruda (pembrolizumab). Indicated for use in melanoma, nonÃÆ'Ã'¢Ã¢ââ'¬Å¡Ã¢â€šâ'¬ÃƒÂ¢Ã¢â€šâ'¬Ã…“small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high (MSI-H) or a mismatch repair deficient (dMMR) solid tumor, gastric or gastroesophageal junction (GEJ) adenocarcinoma, squamous cell carcinoma of the esophagus, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma (MCC), and renal cell carcinoma (RCC).

Patients with mantle cell lymphoma (MCL) have a particularly poor prognosis when treated with standard chemotherapy, with a median overall survival of only 3 years. These patients have therefore been considered for first-line treatment with more aggressive or experimental strategies, such as high-dose therapy (HDT) with autologous stem cell transplantation (ASCT). While high rates of clinical remission have been achieved with HDT/ASCT, this procedure alone is not believed to be curative and different treatment strategies are being developed to improve outcomes in this group of patients. Single-agent rituximab is active in both newly diagnosed and relapsed MCL and therefore the addition of rituximab to chemotherapy regimens and/or HDT/ASCT may enhance their efficacy. Outside the transplantation setting, rituximab plus chemotherapy has been shown to be highly active in MCL, and preliminary data from randomized trials suggest that the combination may yield superior results compared with chemotherapy alone. The addition of rituximab to transplantation protocols appears to be a very promising strategy for patients with MCL, given as an in vivo purge before HDT/ASCT and/or as posttransplant maintenance therapy. Two phase II clinical trials with rituximab given as an in vivo purge during stem cell mobilization and as posttransplant immunotherapy in patients with previously untreated MCL have generated very promising data. Rituximab is an important addition to cytotoxic therapy and with HDT/ASCT represents a highly active therapy that may be superior to conventional treatment or HDT/ASCT alone in MCL. Randomized prospective studies and longer follow-up are needed to determine whether these regimens can achieve longer survival or possibly cure in this disease.

Introduction: Recurrent/refractory diffuse large B cell lymphoma (DLBCL) remains an area of unmet clinical need. Although autologous stem cell transplant (ASCT), and more recently chimeric antigen receptor (CAR) T cell therapy, have made a significant impact, many patients are ineligible for these treatments due to advanced age, comorbidities, or financial or geographical limitations, or they have relapsed following these treatments. Survivin, a novel target in DLBCL, is overexpressed in approximately 60% of untreated DLBCL with higher rates in relapsed disease. DPX-Survivac, a T cell activating therapy, elicits a strong and prolonged immune response against tumors expressing survivin. DPX-Survivac is combined with pembrolizumab, an anti-PD-1 checkpoint inhibitor, and intermittent low dose cyclophosphamide (CPA), used for an immunomodulatory effect. Pre-clinical studies have demonstrated increased efficacy of this DPX-based drug combination in controlling of tumor growth in murine tumor models, resulting in improved survival (Weir et al. J Immunother Cancer 2016). Methods: "SPiReL" is a Phase 2 non-randomized, open label, efficacy and safety study. Subjects with recurrent/refractory DLBCL with confirmed survivin expression are eligible for participation. Participants must also be ineligible for curative therapy. Study treatment includes administering two doses of 0.5 mL of DPX-Survivac 3 weeks apart followed by up to six 0.1 mL doses every 8 weeks. Intermittent low dose cyclophosphamide is administered orally at 50 mg twice daily for 7 days followed by 7 days off. Pembrolizumab 200 mg is administered every 3 weeks. Study participants continue active therapy for up to one year or until disease progression, whichever occurs first. The primary objective is to document the response rate to this treatment combination using modified Cheson criteria. Secondary objectives include duration of response and safety. Exploratory endpoints include T cell response, tumor immune cell infiltration, and gene expression analysis. Enrollment is ongoing with a goal of up to 25 subjects in this national, multi-center study. Trial design is shown below the text. Results: At the time of data cut-off, 23 subjects have been screened and 12 have been enrolled. The demographics of enrolled subjects include: median age is 75.5 years (50-82), with 3 male subjects participating. The median number of prior therapies is 2.5 (1-6), with 4 subjects having previously undergone ASCT. The median time from diagnosis to screening is 31 months (8-151). Of the 12 enrolled subjects, 3 were not evaluable for clinical efficacy due to early disease progression. One subject has not yet reached the first time point for assessment. In the Per Protocol analysis of 8 evaluable subjects ("PP", N = 8); as compared to a Full Analysis Set ("FAS", N = 11); 7 of 8 subjects (PP = 87.5%) demonstrated clinical benefit, including 6 with tumor regressions (FAS = 63.6%); 2 subjects (PP = 25%) achieved a complete response, 1 of whom has completed the 1 year study period (FAS = 18.2%); 3 subjects (PP = 37.5%) had a partial response (FAS = 27.3%); and 3 (PP = 37.5%) had stable disease remaining on study for 4, 6 and 8 months (FAS = 27.3%). This treatment combination is well-tolerated with mostly grade 1 and 2 adverse events reported; a single grade 3 maculopapular rash and 1 case of grade 4 cytopenia has been observed. Of the 23 screened subjects, survivin expression analysis was performed on 18 subjects. All of the 18 samples analyzed were survivin positive, with a range of 60%-100% of lymphoma cells expressing survivin. Preliminary analysis of peripheral blood T cell responses to survivin by ELISPOT assay shows 2 of 4 subjects had strong survivin-specific T cell responses. Both of these subjects had clinical responses. Summary: DPX-Survivac, in combination with pembrolizumab and low dose cyclophosphamide, demonstrates clinical activity and a favorable safety profile in recurrent/refractory DLBCL with 87.5% of evaluable subjects deriving clinical benefit, including 2 complete responses (25%) and 3 partial responses (37.5%) to date. Enrollment is continuing to further explore the synergistic effect of this combination in this population. Figure Disclosures Berinstein: Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Stewart:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda (pemroblizumab) is approved for use to treat Melanoma, Metastatic, Non-Small Cell Lung Cancer, Head and Neck Cancer, Hodgkin's Lymphoma, Urothelial Carcinoma, Gastric Cancer, Cervical Cancer, Hepatocellular Carcinoma, Merkel Cell Carcinoma, Renal Cell Carcinoma, and Small Cell Lung Cancer. In this trial, it is being used to treat Diffuse Large B-Cell Lymphoma.

In the pre-rituximab era, transformation of indolent B-cell lymphoma to diffuse large B-cell lymphoma (DLBCL) was associated with an extremely poor outcome and a median post-transformation survival ranging from 1 to 2 years. We evaluated the impact of rituximab-cyclophosphamide, adriamycin, vincristine, prednisone (R-CHOP) on the survival outcomes of transformed lymphoma compared with de novo DLBCL. Between 2002 and 2010, 317 DLBCL patients who were consecutively diagnosed and treated with R-CHOP were identified at our institution. Patients with transformed lymphoma were included if they had not previously received R-CHOP. Patient characteristics, treatment, and outcome data were retrospectively collected. Sixty patients (19 %) had transformed lymphoma of which 37 (62 %) had transformed from follicular lymphoma, 50 (83 %) were chemotherapy naïve, and 58 (96 %) were rituximab naïve at the time of treatment. With a median follow-up of 31.4 months, 231 patients achieved either complete response or complete response unconfirmed (73 %) with no significant difference between de novo DLBCL (n = 192, 75 %) and the transformed group (n = 39, 65 %) (P = 0.25). Six patients (15 %) relapsed in the transformed group at a median time to relapse of 29.3 months. The 2-year and 5-year overall survivals for all patients were 82 and 72 %, respectively. The overall and progression-free survivals for transformed lymphoma and de novo DLBCL were not statistically different (P = 0.45 and P = 0.38, respectively). With R-CHOP chemotherapy, the prognosis of transformed lymphoma in patients with minimal chemotherapy exposure for indolent disease is similar to that of de novo DLBCL.

Background TRPS-1 is a new GATA transcription factor that is differentially expressed in breast cancer (BC) where it been found recently to regulate epithelial-to-mesenchymal transition (EMT). Patients and methods We carried out a quantitative immunohistochemistry (qIHC) analysis of TRPS-1 expression in 341 primary-stage I–III BC samples in relation to patient clinical characteristics as well as its prognostic value, especially in an estrogen receptor-positive (ER+) subgroup. Results Higher TRPS-1 expression was significantly associated with a number of clinical and pathological characteristics as well as with improved overall survival (OS) and disease-free survival (DFS). Among stage I/II ER+ BC patients who received endocrine therapy alone, those with high TRPS-1 expression had significantly longer OS and DFS. There was also a strong association between TRPS-1 levels and the EMT marker E-cadherin in the ER+ invasive ductal carcinoma cases. Analysis of gene expression data on a panel of BC lines found that TRPS-1 expression was low or absent in BC lines having enriched mesenchymal features. Conclusions Our data indicated that TRPS-1 is an independent prognostic marker in early-stage BC and a new EMT marker that can distinguish patients with ER+ BC who will respond longer to adjuvant endocrine therapy.

Background Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3). Methods Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E749-57 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 μg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α+ peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRβ sequencing using genomic DNA. Results Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α+ TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α+ T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group. Conclusions These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0169-2) contains supplementary material, which is available to authorized users.

Twenty-seven subjects with squamous cell cancer of the head and neck received the neoadjuvant IRX-2 immunotherapy regimen prior to surgery in a Phase 2 trial. Pretreatment tumor biopsies were compared with the primary tumor surgical specimens for lymphocyte infiltration, necrosis and fibrosis, using hematoxylin and eosin stain and immunohistochemistry in 25 subjects. Sections were examined by three pathologists. Relative to pretreatment biopsies, increases in lymphocyte infiltration (LI) were seen using H and E or immunohistochemistry. CD3+ CD4+ T cells and CD20+ B cells were primarily found in the peritumoral stroma and CD3+ CD8+ T cells and CD68+ macrophages were mainly intratumoral. LI in the surgical specimens were associated with reductions in the primary tumor size. Improved survival at 5 years was correlated with high overall LI in the tumor specimens. Neoadjuvant IRX-2 immunotherapy regimen may restore immune responsiveness presumably by mobilizing tumor infiltrating effector lymphocytes and macrophages into the tumor.

7 Background: The IRX-2 biologic is an injectable immunotherapy containing cytokines derived from stimulated lymphocytes. In preclinical models, IRX-2 activates T-cells, natural killer (NK) cells, macrophages, and dendritic cells, and facilitates maturation of antigen-presenting cells. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved outcomes in early stage breast cancer (ESBC), and neoadjuvant IRX-2 increased TILs in a head/neck squamous cell carcinoma trial. We conducted a phase Ib trial to evaluate the feasibility/activity of IRX-2 in ESBC. Methods: Beginning 21 days prior to surgical resection, enrolled operable patients with stage I-III ESBC received the pre-operative IRX-2 regimen consisting of a single low dose of cyclophosphamide (300 mg/m2 to facilitate T-regulatory cell depletion), followed by 10 days of subcutaneous peri-areolar injections of IRX-2 into the affected breast (1 mL × 2 at tumor axis and at 90°). Endpoints were feasibility (primary), sTIL count (secondary), and immune monitoring by flow cytometry and immune transcriptome analysis (Nanostring PanCancer Immune Panel). Results: As of October 2017, 12 patients are enrolled and evaluable. All patients received all planned injections with no treatment-related surgical delays, complications, or grade III/IV toxicities. Treatment was associated with a mean 52% relative increase in sTILs (range –25% to +166%, p = 0.02). RNA expression profiling revealed the greatest increases in Th1 cell signature (mean +28.5%, range -5.3% to +283%), but also increases in CD8+, NK, and DC signatures. PDL1 RNA expression increased in 10/12 patients (mean +131.5%, range –83% to +1112%). In blood, increases in T-cell activation markers (ICOS, HLADR, and CD38) and T-reg depletion were observed at 1-month follow-up. Conclusions: Peri-lymphatic IRX-2 was well tolerated with preliminary evidence of sTIL recruitment, PDL1 upregulation, and peripheral lymphocyte activation. Study enrollment is ongoing, including patients for a pre-neoadjuvant-chemotherapy triple-negative ESBC cohort. Clinical trial information: NCT02950259.

Immunotherapy employs cytokines for modifying local inflammatory reactions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate dendritic cells, macrophages, and granulocytes leading to clinical trials using GM-CSF-based cancer vaccine approaches. Interleukin-2 (IL-2) is an important T cell stimulatory cytokine approved as exogenous antitumor agent. The ALVAC viral vector system uses a recombinant canarypox virus for local gene expression. We report a phase I clinical trial using intratumoral administration of ALVAC GM-CSF or ALVAC IL-2 in skin metastases of melanoma or leiomyosarcoma. ALVAC GM-CSF and ALVAC IL-2 were injected at 107.12 and 106.92, 50% cell culture infectious dose in eight metastases with acceptable tolerability. Local and systemic inflammatory reactions were observed. The transgene determined the local infiltrate: GM-CSF induced monocyte and macrophage enrichment of the peritumoral inflammatory infiltrate, whereas IL-2 increased local T lymphocytes. Stable disease of injected lesions was seen after ALVAC GM-CSF application, whereas ALVAC IL-2 treatment led to partial regression in three out of eight injected tumors, accompanied by decreased expression of melanocytic antigens. Local GM-CSF expression could be induced. In summary, ALVAC GM-CSF and ALVAC IL-2 injections are safe and can mediate local biologic and immunologic effects.

Follicular lymphoma (FL) is a unique disease characterised by a long natural history and responsiveness to many different therapies. We have reviewed the prognostic significance of the quality of both clinical and molecular responses for patients with FL. We have found that, as might be expected, patients who achieve a complete clinical response to treatment have a better prognosis than patients who achieve an incomplete or a partial response. However, unlike aggressive lymphomas, treatments that produce a higher frequency of complete responses do not result in better survival outcomes than treatments that produce lower complete response rates. Recent improvements in technologies have enabled quantitative monitoring of responses at the molecular level and at much higher degrees of sensitivity than can be obtained at the clinical level. Although these data are very heterogeneous and have many limitations, data are emerging that demonstrate that achieving molecular remissions after standard dose chemotherapy, high dose chemotherapy or various immunotherapies may have prognostic significance for patients with FL. With sensitive, quantitative, standardised and reproducible tools for molecular monitoring and with the combination of novel targeted biological therapies, we are approaching an era, where the potential to cure patients with FL is being turned into a reality.

Recently, immunotherapy has been widely investigated for the treatment of cancer. While much of the success in this area lies with passive immunotherapy, the focus of this review is active vaccination strategies, which are showing promise. Cancer cells express a wide profile of different proteins, some of which are related to oncogenic transformation and are specific to cancer cells only. However, in cancer, most protein targets presented to the immune system from tumor cells are self-antigens. The immune system is highly tolerant of, and therefore does not react to, these self-antigens. Active immunotherapy aims to reverse this immune tolerance so the immune system can respond appropriately to self-antigens. To generate a successful antitumor response, several features should be considered: a target antigen on tumor cells to direct the immune response, a platform to present the vaccine-derived antigen to the immune system, an adjuvant to enhance immune stimulation, and appropriate monitoring techniques. New tools are becoming available for monitoring, although the clinical relevance for these assays needs to be established. Optimism for cancer vaccine approaches are increasing. Recent studies have shown that it is possible to break immune tolerance to self-antigens. Promising therapeutic clinical activity has been demonstrated, and other studies have shown that cancer vaccines are most beneficial for minimal residual disease states. Further optimization of cancer vaccines and larger clinical studies are required.

High-dose therapy with stem-cell transplantation is a potentially curative therapy for younger patients with relapsed aggressive non-Hodgkin's lymphoma (NHL) and is also under investigation in relapsed indolent NHL. There are, however, risks associated with this treatment strategy. Autologous stem-cell transplantation (ASCT) continues to be associated with a high risk of relapse, while graft-versus-host disease is a major limiting factor with allogeneic stem-cell transplantation. The presence of minimal residual disease (MRD) in the harvested, re-infused stem cells, or remaining in the patient following chemotherapy, is associated with relapse after ASCT. As a result, monitoring and eradicating MRD has become a major focus of many studies in NHL. Rearrangement and overexpression of the bcl-1 and bcl-2 genes are the hallmarks of mantle-cell and follicular lymphoma, respectively, and evidence suggests that they are promising surrogate markers of MRD. Polymerase chain reaction analysis is a sensitive methodology used to monitor the status of occult lymphoma cells bearing these genetic aberrations, and results from trials of ASCT have shown that clearance of bcl-1/JH- and bcl-2/JH-positive cells following treatment is associated with a significant improvement in outcome. Rituximab, the anti-CD20 monoclonal antibody, is increasingly used for in vivo purging and can effectively eradicate bcl-1/JH- and bcl-2-positive cells. If the encouraging preliminary results with rituximab are maintained with a longer follow-up, this agent could play a pivotal role in improving outcome after stem-cell transplantation in NHL.

In clinical trials, metronomic cyclophosphamide (CPA) is increasingly being combined with vaccines to reduce tumor-induced immune suppression. Previous strategies to modulate the immune system during vaccination have involved continuous administration of low dose chemotherapy, studies that have posed unique considerations for clinical trial design. Here, we evaluated metronomic CPA in combination with a peptide vaccine targeting HPV16E7 in an HPV16-induced tumor model, focusing on the cytotoxic T-cell response and timing of low dose metronomic CPA (mCPA) treatment relative to vaccination. Mice bearing C3 tumors were given metronomic CPA on alternating weeks in combination with immunization with a DepoVax vaccine containing HPV16E749–57 peptide antigen every 3 weeks. Only the combination therapy provided significant long-term control of tumor growth. The efficacy of the vaccine was uncompromised if given at the beginning or end of a cycle of metronomic CPA. Metronomic CPA had a pronounced lymphodepletive effect on the vaccine draining lymph node, yet did not reduce the development of antigen-specific CD8+ T cells induced by vaccination. This enrichment correlated with increased cytotoxic activity in the spleen and increased expression of cytotoxic gene signatures in the tumor. Immunity could be passively transferred through CD8+ T cells isolated from tumor-bearing mice treated with the combinatorial treatment regimen. A comprehensive survey of splenocytes indicated that metronomic CPA, in the absence of vaccination, induced transient lymphodepletion marked by a selective expansion of myeloid-derived suppressor cells. These results provide important insights into the multiple mechanisms of metronomic CPA induced immune modulation in the context of a peptide cancer vaccine that may be translated into more effective clinical trial designs.

Purpose Idiotypes (Ids), the unique portions of tumor immunoglobulins, can serve as targets for passive and active immunotherapies for lymphoma. We performed a multicenter, randomized trial comparing a specific vaccine (MyVax), comprising Id chemically coupled to keyhole limpet hemocyanin (KLH) plus granulocyte macrophage colony-stimulating factor (GM-CSF) to a control immunotherapy with KLH plus GM-CSF. Patients and Methods Patients with previously untreated advanced-stage follicular lymphoma (FL) received eight cycles of chemotherapy with cyclophosphamide, vincristine, and prednisone. Those achieving sustained partial or complete remission (n = 287 [44%]) were randomly assigned at a ratio of 2:1 to receive one injection per month for 7 months of MyVax or control immunotherapy. Anti-Id antibody responses (humoral immune responses [IRs]) were measured before each immunization. The primary end point was progression-free survival (PFS). Secondary end points included IR and time to subsequent antilymphoma therapy. Results At a median follow-up of 58 months, no significant difference was observed in either PFS or time to next therapy between the two arms. In the MyVax group (n = 195), anti-Id IRs were observed in 41% of patients, with a median PFS of 40 months, significantly exceeding the median PFS observed in patients without such Id-induced IRs and in those receiving control immunotherapy. Conclusion This trial failed to demonstrate clinical benefit of specific immunotherapy. The subset of vaccinated patients mounting specific anti-Id responses had superior outcomes. Whether this reflects a therapeutic benefit or is a marker for more favorable underlying prognosis requires further study.

Treatment outcomes were documented for 204 adult patients with clinical Stage I-II Hodgkin's disease who were treated with risk-adapted ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) and radiotherapy (RT) at the Toronto-Sunnybrook Regional Cancer Centre between 1984 and 1994. Forty-nine patients with clinical Stage I disease (excluding bulky mediastinal presentations) and 50 patients with a combination of clinical Stage IIA disease, age 50 years or less, and favourable pathology (lymphocyte predominant or nodular sclerosing histology) were identified as low risk and treated with RT alone to 35 Gy. One hundred and five high-risk patients were treated with chemotherapy (86 with ABVD) followed by RT to 25 Gy. The 7-year cause-specific, overall and disease-free survivals were 95%, 90% and 75% respectively for the low-risk cohort, and 91%, 90% and 88% respectively for the high-risk cohort. In-field relapses accounted for 50% of the failures in both groups. Sixteen of 24 (67%) patients with RT failure and 6/14 (43%) with combined modality therapy (CMT) failure were salvaged. Twenty-eight per cent of the patients treated with RT and 21% of those treated with CMT developed hypothyroidism by 7 years. Fatal complications were recorded in 6% of the low-risk patients managed with RT and 8% of high-risk patients managed with CMT. Septic death and second malignancy accounted for the majority of treatment-related fatalities. Risk-adapted therapy emphasizing RT alone for selected patients with favourable prognostic factors and CMT based on ABVD provides excellent long-term disease control. Further treatment refinements, including the wider application of CMT with lower doses of chemotherapy and RT, will be required to reduce the rate of fatal complications to more acceptable levels.

This study was designed to evaluate the efficacy and toxicity of dose intensifying DHAP (dexamethasone, cytarabine and cisplatin) salvage chemotherapy by adding mitoxantrone with GM-GSF support in patients with relapsed or refractory non-Hodgkin's lymphoma (NHL). From March 1992 to January 1995, 22 patients with intermediate and high grade (aggressive) NHL refractory or relapsed after adriamycin containing chemotherapy regimens were treated with M-DHAP+GM-CSF, (dexamethasone 40 mg i.v. days 1-4, cisplatin 100 mg/m2 i.v. by continuous infusion over 24 hours on day 1, cytarabine 2 gm/m2, i.v. every 12 hours for 2 doses on day 2, mitoxantrone 10 mg/m2 i.v. on days 3 and 4 and GM-CSF 250-500 microg/m2 s.c. daily beginning day 5 until absolute neutrophil count recovery. Most patients had poor prognostic factors including primary refractory disease (18/22), bulky disease (12/22), elevated LDH (9/22), or bone marrow involvement (8/22). All 22 patients were evaluable. The overall response rate was 41% (CR 23% and PR 18%). There were three toxic deaths, all related to sepsis. Median progression free survival (PFS) and overall survival (OS) rates were 5.2 months and 11.8 months respectively. At the same time of the analysis two patients were alive after high-dose therapy and bone marrow transplant at 34 and 36 months follow-up and two were alive with disease. The maximal acceptable dosage of mitoxantrone was 10 mg/m2 x 2 due to serious hematologic toxicity. Treatment delays and dose reductions compromised delivering the optimal dose intensity of M-DHAP. A poor prognostic group of patients with refractory or recurrent aggressive lymphoma, many of whom were not eligible for high-dose therapy and stem cell transplantation were treated with repeated cycles of dose intensified DHAP with growth factor support. Although M-DHAP had therapeutic activity even in patients considered to have primary refractory disease, myelosuppression was dose limiting and frequently limited the number of cycles. Therefore, if M-DHAP is to be further evaluated, therapeutic results may be improved further by incorporating strategies to reduce myelotoxicity such as the use of growth factors to reduce platelet transfusion requirements or the use of autologous stem cell support after each cycle.

Because of evidence that failure to deliver full dose CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) may compromise the outcome of elderly patients with aggressive non-Hodgkin's lymphoma (NHL), we attempted to deliver full dose CHOP to these patients. The objective of this review was to assess the relative received dose intensity (ARRDI), toxicity and outcome of elderly patients treated with curative intent with CHOP at our centre. Charts were reviewed of all patientsor = 65 years with newly diagnosed aggressive NHL referred to the Toronto-Sunnybrook Regional Cancer Centre (TSRCC) for initial management from 1990-1995 before routine use of G-CSF. Sixty eligible patients were identified. 31 received CHOP +/- radiation (XRT), 9 other curative treatment and 20, palliative treatment. The mean ARRDI calculated on 29/31 patients receiving CHOP was .86; 41%=1.0, 24%=.90-.99, 14%=.75-.89 and 21%=.75. During 141 cycles of CHOP. 17 (12%) episodes of febrile neutropenia (FN) occurred in 14 (45%) patients and other grade 3/4 toxicity occurred in10% of patients. There were 3 (10%) toxic deaths. Sixteen (52%) patients required a total of 29 admissions to hospital for FN (59%) or other causes. Of the 31 patients, 16 (52%) achieved a complete remission (CR), 7 (23%) a partial remission-1 (PR-1), 2 (6%) a partial remission-2 (PR-2), 1 (3%) had no response (NR), 2 (6%) had progressive disease and 3 (10%) were not evaluable (NE). The median progression free survival (PFS) and overall survival (OS) were (16+) months and (24.5) months respectively. We found that physician biases resulted in the selection of; younger patients (median 71 vs. 80 years), patients with a better ECOG performance status (or =2, 13% vs. 50%) and patients with less co-morbid illness (42% vs. 90%) for attempt at curative treatment with CHOP chemotherapy. Age was never the sole reason for offering palliative treatment. In conclusion, a subset of patients over the age of 65 with aggressive NHL, who have a good performance status and minimal co-morbid illness can tolerate full dose CHOP chemotherapy without G-CSF support. Future strategies should emphasize full dose treatment with curative intent with minimization of both hematologic and non-hematologic toxicity. Clinical studies are required to determine whether routine G-CSF support will reduce toxicity or improve outcome in this group of patients.

Summary Background Monoclonal antibodies are being utilized for treatment of patients with low-grade non-Hodgkin’s lymphoma as well as other cancers. Results from phase I and II clinical studies has shown that the chimeric monoclonal antibody Rituximab has minimal toxicity and significant therapeutic activity in low grade non-Hodgkin’s lymphoma. Patients and methods We have recently reported on a multicentre pivotal phase III clinical trial involving 166 patients with recurrent low-grade lymphoma who were treated with four infusions of Rituximab. Eighty patients (48%) achieved objective responses including 10 patients (6%) with complete responses. Overall, 126 patients (76%) had a ≥ 20% reduction in overall tumor size. The median response duration and time to progression are 11.6 and 13.2 months, respectively. The infusional and long term toxicities were limited. Results In this report we describe the pharmacokinetic data obtained on these patients. Measurable concentrations of Rituximab were detected in all patients after the first infusion and increased throughout the treatment course. The half-life of the monoclonal antibody increased from 76.3 hours after the first infusion to 205.8 hours after the fourth infusion and was concomitant with a four-fold decrease in the antibody clearance. At three months and six months post-treatment, the median Rituximab serum levels were 20.3 μg/ml (range 0.0 to 96.8 μg/ml in 104 patients) and 1.3 μg/ml (range 0.0–28.7 μg/ml in 13 patients), respectively. A statistically significant correlation was found between the median antibody concentration and response for multiple time points during the treatment and followup. The mean serum antibody concentration was also inversely correlated with measurements of tumor bulk and with the number of circulating B cells at baseline. Conclusions We conclude that Rituximab is therapeutically effective against B-cell lymphoma. Pharmacokinetic data suggests that certain subsets of patients may possibly benefit from increased dosing and studies to address this are currently underway.

Follicular lymphoma (FL) is characterized by an initial response to treatment with inevitable relapse. We evaluated chemoimmunotherapy resistance (CIR resistance) including transformation. We identified patients who received rituximab combination therapy for symptomatic FL. CIR resistance was defined as disease progression during rituximab-based chemoimmunotherapy, rituximab maintenance or within 6 months of treatment completion. Our primary outcome was time to early progression (CIR resistance). Between July 2006 and April 2010, 132 patients met the inclusion criteria and 22 (16.7%) demonstrated CIR resistance with a median follow-up of 33 months. High-risk Follicular Lymphoma International Prognostic Index (FLIPI) score was predictive of CIR resistance (hazard ratio [HR] 2.43; 95% confidence interval [CI], 1.4-4.1; p = 0.001). Overall, eight patients (36.3%) transformed (biopsy-proven), with no transformation in the chemoimmunotherapy responder group. Median overall survival in the CIR resistant group was 47 months. Patients with CIR resistance had high rates of histologic transformation and shorter survival with poor response to next therapy.

We have studied, as part of a group of international multicenter phase II clinical trials, the toxicity and effectiveness of CAMPATH1H administered intravenously three times a week in an outpatient setting to patients with recurrent or progressive low grade lymphoma. We report here on the toxicity and therapeutic results of the first seven patients treated before the study was closed prematurely because of unacceptable toxicity. Classical complete or partial responses of treatment were seen in three of seven patients. One complete response lasted 8.5 months and the other complete response is ongoing at 1 year. Responses occurred in nodal sites as well as in skin and peripheral blood. The first three or four antibody infusions in each patient was associated with grade 1 or 2 side-effects including rigor, fever, facial flushing, nausea, vomiting, hives, wheezes, hypotension, and/or diarrhea but these subsequently decreased or disappeared. The most significant toxicity was profound lymphopenia and associated infection, usually viral. Six of seven patients had culture or serologically documented infections and four patients had two or more such episodes. All infections responded to temporary discontinuation of antibody therapy and appropriate antiviral or antibiotic agents. We conclude that CAMPATH1H monoclonal antibody has therapeutic activity against low grade non-Hodgkin's lymphoma but that this activity is limited by marked lymphopenia and an unacceptably high frequency of serious infection at the dose and schedule used in this trial.

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. Using a HPV-expressing murine tumor model (C3), we found that treatment with mCPA (20 mg/kg/day PO) in combination with a DPX peptide vaccine caused selective enrichment of antigen-specific CD8+ T cells, resulting in increased immune responses detected by IFN-ã ELISPOT and in vivo cytotoxicity assay. The combination provided long term-control of tumors when initiated within one week of tumor implantation, however efficacy was limited in mice bearing advanced tumors. Antigen-specific CD8+ T cells could be detected infiltrating advanced tumors by flow cytometry, along with increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and PD-1 blockade (with anti-PD-1 or anti-PD-L1) resulted in tumor control of established tumors which were not successfully treated with antibody monotherapy. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRâ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRâ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRâ sequences (p Citation Format: Genevieve Weir, Olga Hrytsenko, Tara Quinton, Mohan Karkada, Neil L. Berinstein, Marianne Stanford, Marc Mansour. Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T 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 4903.

e14578Background: Survivin is strongly expressed in tumors of about 60% of diffuse large B-cell lymphoma (DLBCL) patients. DPX-Survivac contains survivin HLA class I peptides in a novel formulation...

e14577Background: Survivin is an important protein that regulates apoptosis, proliferation, and angiogenesis of tumors. Increased expression of survivin in various tumors correlates with progressio...

PURPOSE The purpose of this review was to assess the outcome of patients with non-Hodgkin's lymphoma (NHL) who achieve an incomplete or slow response to front-line chemotherapy and to determine whether salvage treatment with intensive combination chemotherapy with or without autologous bone marrow transplantation (ABMT) is successful in such patients. METHODS A comprehensive literature search of studies using combination chemotherapy for the front-line therapy of advanced-stage intermediate- and high-grade NHL and for salvage therapy of patients with a partial response (PR) was reviewed. RESULTS The median survival duration of patients with a PR ranged between 5 to 14 months, while the median survival duration of patients with a complete response (CR) was not reached in many studies. For patients in CR, the probability of survival at 24 months ranged between 0.79 to 1, while for patients in PR it ranged from 0 to 0.31. The rapidity of a response to front-line therapy was often found to be of prognostic importance. Patients who relapsed after a PR to front-line therapy had similar outcomes to intensive salvage therapy as those who relapsed after a CR. ABMT performed immediately after a PR to induction therapy, before progressive disease occurred, resulted in high CR rates in nonrandomized studies. CONCLUSION Patients with aggressive NHL who experience a PR or who respond slowly to front-line chemotherapy have a poor prognosis. Early introduction of dose-intensive salvage therapy before the development of progressive disease may benefit patients with a PR and requires testing in randomized clinical trials.

In order to study mechanisms of immunoglobulin somatic variation in committed immunoglobulin (Ig) expressing B cells, we used the fluorescent activated cell sorter to isolate rare variants from a surface Ig positive (sIg+) diffuse large cell B lymphoma cell line (mu lambda+). These variants were either negative for sIg expression (sIg-) or expressed sIg which differed from the original parental tumor cell line, both in idiotypes and Ig lambda isotypes (sIg+Id-). In the following report we review the results from the studies of these variants. DNA analysis showed that all variants had new Ig lambda gene rearrangements, which had occurred either on a previously excluded allele, or on the productively rearranged allele of the parental cell line. The sIg- variants, which had undergone nonfunctional Ig lambda rearrangements on the expressed parental allele, and thereby deleted the productive rearrangement, continued to functionally rearrange the same allele and regenerated sIg expression. While the parental cell line expressed low levels of the recombination activating genes, RAG1 and RAG2, expression of these genes were considerably upregulated in both the immunoglobulin negative and the idiotypic variants. Somatic immunoglobulin V gene hypermutation did not contribute to the observed immunoglobulin somatic variation. These results demonstrate that, through differential expression of the RAG genes, sIg+ B cells are able to somatically alter their sIg receptors through secondary Ig lambda gene rearrangements. This mechanism may allow B cells with non-selectable, or auto-reactive, antigen receptors to alter these receptors (receptor editing). Ongoing Ig gene rearrangement may limit the usefulness of immunotherapeutic approaches directed at the antigen receptor in some diffuse large cell lymphomas.

IRX-2, a primary cell-derived biologic with pleotropic immune activity, was shown to induce increased lymphocyte infiltrations into the tumor of patients with head and neck squamous cell cancer (HNSCC) after 10 days of neoadjuvant therapy (Berinstein et al. 2011). In the same patients enrolled in the Phase II study, peripheral blood lymphocyte subsets were monitored pre- and post-IRX-2 therapy to evaluate changes induced by IRX-2.Absolute lymphocyte numbers were determined in whole blood using the TetraONE System. Lymphocytes were further separated on Ficoll-Hypaque gradients and evaluated by multiparameter flow cytometry. Lymphocyte numbers, including regulatory T cells (Treg) and naïve, memory and effector T cells, were compared in pre- and post-therapy specimens.Total lymphocyte numbers remained unchanged after IRX-2 therapy. Significant changes occurred in numbers of circulating B cells and NKT cells, which decreased following IRX-2 therapy. The frequency of circulating Treg (CD4(+)CD25(high)) remained unaltered (e.g., 6.7 ± 0.6% vs. 7.5 ± 0.8%; means ± SEM) as was the CD8(+)/Treg ratio (6.6 before and 6.7 after IRX-2 therapy). The mean absolute number of CD3(+)CD45RA(+)CCR7(+) (naïve) T cells was decreased after IRX-2 therapy but numbers of total memory (i.e., central and peripheral) and terminally differentiated T cells were unchanged.IRX-2-mediated reductions in B and NKT cell numbers in the blood suggest a redistribution of these cells to tissues. A decrease in naïve T cells implies their up-regulated differentiation to memory T cells. Unchanged Treg numbers after IRX-2 therapy indicate that IRX-2 does not expand this compartment, potentially benefiting anti-tumor immune responses.

The trichorhinophalangeal syndrome 1 (TRPS-1) gene is a novel GATA transcription factor family member. Previously, using a gene expression profiling and immunohistochemistry (IHC) screen, we identified TRPS-1 as a highly prevalent gene in breast cancer (BC), expressed in >90% of estrogen receptor alpha (ERα)+ and ERα− BC subtypes. TRPS-1 was also shown to be expressed in prostate cancer where it was shown to play a proapoptotic function during androgen withdrawal possibly through regulating antioxidant metabolism. The role of TRPS-1 and its prognostic significance in hormone-dependent and hormone-independent BC however is not known. In this study, we developed a new quantitative IHC (qIHC) method to further study TRPS-1 as a marker and possible prognostic indicator in BC. By using this method, a quantitative parameter for TRPS-1 expression called a quick score (QS) was derived from the measured labeling index and mean optical density after IHC and applied to a set of 152 stage II/III BC patients from 1993 to 2006 who did not receive preoperative chemotherapy. Associations between QS and tumor characteristics were evaluated using the Kruskal–Wallis test. A wide range of TRPS-1 QS was found among the sample set with higher TRPS-1 QS significantly associated with tumor ERα (p = 0.023 for QS and p = 0.028 for Allred score), progesterone receptor (p = 0.009), and GATA-3 (p

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide-induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. In syngeneic murine models, mCPA on alternating weeks (20 mg/kg/day PO) in combination with DPX peptide vaccines provided long-term control of established tumors. Using a HPV-expressing tumor model (C3), we found that the combination of mCPA with vaccination caused selective enrichment of antigen-specific CD8+ T cells resulting in increased immune responses detected by IFN-γ ELISPOT and in vivo cytotoxicity assay, as well as improved protection from tumors. Efficacy of the vaccine and mCPA combination was limited in mice bearing advanced tumors. However, antigen-specific CD8+ T cells could be detected infiltrating the tumor by flow cytometry along with increased expression of PD-1 by qPCR, suggesting that the combination therapy was able to generate strong cytotoxic T cell response but was still subject to tumor induced suppression within the tumor microenvironment. Tumor bearing mice treated with vaccine, mCPA and anti-PD-1 blockade (with anti-PD1 or anti-PDL1) could control and induce regression of established tumors which could not be successfully treated with antibody monotherapy. Ex vivo analysis of tumor infiltrating cells demonstrated that anti-PD1 treatment did not further enhance tumor infiltration with antigen-specific CD8 T cells induced by the vaccine/ mCPA treatment, however, RT-qPCR analysis of the tumor demonstrated an increase in expression of cytotoxic T cell gene signatures with anti-PD1 treatment within the tumor; these results were confirmed in RT-qPCR analysis of tumor draining lymph node. An increased expression of PDL-1 was also detected in all vaccinated mice, regardless of whether they were also treated with anti-PD1. We conclude that enhanced tumor control mediated by the tri-therapy is mediated by highly active, tumor-specific cytotoxic T lymphocytes produced by a strongly immunogenic vaccine. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac. Citation Format: Genevieve Weir, Olga Hrytsenko, Marianne Stanford, Mohan Karkada, Neil Berinstein, Marc Mansour. Anti-PD1 blockade increases the activity of tumor infiltrating antigen-specific CD8 T cells induced by a potent peptide vaccine in combination with metronomic cyclophosphamide. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B91.

Survivin, a member of the inhibitors of the apoptosis protein family, is involved in critical pathways of cancer cell growth and survival. Survivin overexpression is detected in 90% of epithelial ovarian cancers. DPX-Survivac is a peptide vaccine containing five survivin HLA class I peptides (A1, A2, A3, A24 and B7) and a universal HLA class II helper peptide derived from tetanus toxin (A16L) formulated in the novel adjuvanting vaccine platform DepoVaxTM. In a phase I clinical study DPX-Survivac was combined with a continuous (metronomic) low dose of cyclophosphamide (mCPA) to treat patients with advanced or recurrent ovarian cancer. In this study, we found mCPA significantly enhanced the immune responses induced by DPX-Survivac. In order to investigate this effect and to understand the mechanisms involved, we developed an ovarian tumor model in HLA-A2 transgenic mice. Vaccination of HLA-A2 transgenic mice generates an immune response towards the A2-restricted survivin peptide in DPX-Survivac. Mouse Ovarian Surface Epithelial (MOSE) cell lines are “natural” ovarian cancer models established by spontaneous in vitro transformation of primary ovarian surface epithelial cells. MOSE cell lines were confirmed to express HLA-A2 and overexpress survivin, and formed subcutaneous and intraperitoneal ovarian tumors when implanted into syngeneic recipients. HLA-A2 transgenic mice bearing MOSE tumors were treated with either DPX-Survivac, mCPA (administrated at 20 mg/kg/day in drinking water) or a combination of mCPA and DPX-Survivac. We found that combining DPX-Survivac with mCPA provided most effective control of tumor growth. Ex vivo IFN-γ ELISPOT revealed that mCPA increases immunogenicity of the DPX-Survivac in mice bearing MOSE tumors. Analysis of the blood and tumor microenvironment by RT-qPCR showed that mice treated with the combination therapy had an altered immune gene signature with increased cytotoxic T lymphocyte markers compared to mice treated with DPX-Survivac or metronomic cyclophosphamide alone. Our data indicates that the combination treatment enhances the infiltration of CD8+ T cells into tumors, enhancing tumor specific immune responses. In summary, we have established a new model of epithelial ovarian cancer in HLA-A2 transgenic mice. This model was used to investigate the immunogenicity and efficacy of DPX-Survivac, an HLA-restricted peptide vaccine with proven immunogenicity in human clinical trials, in combination with the immune modulator mCPA. This model can be used to explore mechanisms of establishment and progression of ovarian cancer as well as for evaluation of new treatment strategies including new immune modulator/ vaccine combinations. Citation Format: Olga Hrytsenko, Genevieve M. Weir, Mohan Karkada, Neil L. Berinstein, Marianne M. Stanford, Marc Mansour. Translational study of DPX-survivac peptide vaccine and metronomic cyclophosphamide using a novel HLA-A2/ survivin expressing ovarian tumor model to mimic phase I clinical data [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1415.

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In a phase I clinical trial, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We emulated these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation and explore additional combinations to enhance the therapeutic effect. In syngeneic murine models for ovarian cancer (MOSE), melanoma (B16-F10) and HPV-induced cancer (C3), metronomic low dose cyclophosphamide (mCPA) on alternating weeks (20 mg/kg/day PO) in combination with a DPX peptide vaccine provided long-term control of established tumors. The addition of mCPA caused selective enrichment of antigen-specific CD8+ T cells resulting in increased immune responses detected by IFN-γ ELISPOT and in vivo cytotoxicity assay, as well as improved protection from tumors. Efficacy of the vaccine and mCPA combination was limited in mice bearing advanced tumors, yet antigen-specific CD8+ T cells could be detected infiltrating the tumor by flow cytometry along with increased expression of PD-1 by qPCR, suggesting that the combination therapy was able to generate strong cytotoxic T cell response but was still subject to tumor induced suppression within the tumor microenvironment. A triple combination therapy of mCPA, vaccine and anti-PD1 enabled the control and/or regression of established later-treated tumors which could not be achieved with a combination of mCPA and anti-PD1. To better understand the mechanism of the enhanced protection observed with the tri-therapy, mice were implanted with a mixture of C3 and B16 tumor cells and then treated with a vaccine against the C3 tumors alone, in combination with mCPA and anti-PD1. Splenocytes of mice treated with the tri-therapy produced IFN-γ ELISPOT responses towards both cell lines, suggesting epitope spreading. This effect could not be detected in tumor bearing mice treated with mCPA anti-PD1 without vaccine. We conclude that enhanced tumor control mediated by the tri-therapy is mediated by highly active, tumor-specific cytotoxic T lymphocytes produced by a strongly immunogenic vaccine, resulting in epitope spreading that can further facilitate tumor rejection or control. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac targeting the tumor associated antigen survivin. Citation Format: Genevieve Weir, Olga Hrytsenko, Marianne Stanford, Mohan Karkada, Neil Berinstein, Marc Mansour. Multi-modal treatment with peptide vaccine, metronomic cyclophosphamide and anti-PD1 monoclonal antibody provides effective control of tumors in multiple models. [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 2508. doi:10.1158/1538-7445.AM2015-2508

3072 Background: Survivin is important in apoptosis, proliferation, and angiogenesis. High expression has been linked to progression and drug resistance. DPX-Survivac vaccine (DPX) contains a mix o...

Although follicular non-Hodgkin's lymphoma may respond well to induction therapy, almost all patients will eventually relapse. To reduce the frequency of relapses and prolong disease-free intervals, maintenance treatment may be given to patients who respond or have stable disease after induction therapy. Maintenance treatment is given at regular intervals over prolonged periods in patients who may be asymptomatic, and so it is important that administration is simple, toxicity is minimal and that there is clear evidence of efficacy. The monoclonal antibody rituximab has many characteristics essential for a maintenance therapy: it is both effective and very well tolerated, and can maintain active concentrations in the blood with intervals of several months between administrations, enabling patients to continue with normal life while on maintenance therapy. Randomized trials of rituximab maintenance therapy have confirmed that remissions can be prolonged, and in one randomized trial, early results suggest a survival benefit. The optimal schedule for maintenance therapy has not yet been defined.

Molecular remission in the autograft and bone marrow after transplant are predictive of durable clinical remission in relapsed follicular lymphoma. Thus, a simple reliable method to quantify minimal residual disease (MRD) would improve prognostication in these patients. Fluorescent hybridization probes have been used in real-time quantitative polymerase chain reaction (RQ-PCR) to monitor MRD with a reproducible sensitivity of 0.01%; however, these techniques are expensive and require additional experiments to examine clonality. We describe a SYBR Green I detection method that is more universal, checks clonal identity, yields the same sensitivity for monitoring MRD, and is more economically attractive. Using this method to follow 14 follicular lymphoma patients treated with autologous stem cell transplantation, molecular markers were successfully defined for 12 patients. Median contamination of stem-cell grafts was 0.1% (range, 0 to 13%). Six patients with measurable graft contamination became PCR-negative in blood and bone marrow within 12 months after autologous stem cell transplantation. Three patients free of disease progression (median follow-up of 75 months) are in molecular remission. Increasing fractions of RQ-PCR-positive blood and bone marrow cells reliably predicted morphological and clinical relapse. In one case, both clinical relapse and spontaneous regression were reflected by changes in MRD levels. Thus, our RQ-PCR method reproducibly distinguishes different levels of MRD.

BACKGROUND The route of administration and extent of helper T-cell activation are factors that are likely to be important for the development of effective cancer vaccines. In order to optimize CD8+ cytotoxic T-lymphocyte (CTL) responses, the immunologic effects of direct lymph node (LN) injections of canary pox virus (ALVAC) vectors (expressing the melanoma antigen, gp100) and immunogenic gp100 peptides, along with concomitant injections of the helper adjuvant, tetanus toxoid, were studied in high-risk HLA-A*0201+ patients. METHODS Forty-two patients were vaccinated using six different protocols. Twenty-three patients were ‘primed’ with ALVAC(2)-gp100m and ‘boosted’ with gp100 peptides, either subcutaneously or into an LN. Intranodal (IN) peptides, alone, were administered to six patients. Thirteen patients were given tetanus toxoid initially, and with each gp100 vaccination. Toxicity was recorded and immunologic responses were determined in 35 patients by enzyme-linked immunospot (ELISPOT) and gp100-tetramer binding assays and anti-ALVAC(2) enzyme-linked immunosorbent assays (ELISAs). RESULTS All vaccine protocols were tolerated well. Using stringent criteria for immunologic response, 8 of 18 patients responded to the viral vaccines, in striking contrast to peptides only (0 of 6 patients) or with help in trans from tetanus-reactive T-cells (1 of 11 patients). Changes in gp100-reactive CTL frequencies and ALVAC antibodies were greatest when viruses were injected directly into LNs. CONCLUSIONS IN injections of ALVAC(2)-gp100m viruses are feasible, safe, and may be a superior method of vaccination in humans. CTL responses to this vaccine were not enhanced by tetanus toxoid. Cancer 2006. © 2006 American Cancer Society.

Eighty-five percent of follicular lymphomas possess a characteristic t(14;18) translocation that results in the deregulated expression of the proto-oncogene BCL-2. BCL-2 overexpression alone is insufficient for full cellular transformation and at least 1 other genetic event is believed to be necessary for follicular lymphoma development. Deregulated c-Myc expression has previously been shown to cooperate with Bcl-2 to transform murine fibroblast cell lines and lead to tumor development in mice. We have developed a human model system to study early transformation in lymphoid cells using immortalized lymphoblastoid cells. We sequentially introduced BCL-2 and c-MYC, 2 proto-oncogenes known to be involved in the transformation of B cells into Epstein-Barr virus (EBV)-immortalized human B cells. We show that the c-Myc and Bcl-2 overexpression, together with EBV immortalization were insufficient to cause full cellular transformation as measured by cell proliferation rates, soft agar and tumorigenicity assays. These results show that more than 3 genetic hits (EBV infection, Bcl-2 and c-Myc overexpression) were required for the full cellular transformation of human lymphoblastoid cells. However, subtle changes in cellular proliferation and sensitivity to apoptosis were documented, at non-limiting dilutions. These changes may confer a susceptibility to the modified cells such that they are more susceptible to the acquisition of additional genetic changes and evolve towards a fully transformed state. In addition, the model system developed may be suitable for the identification of further known and novel oncogenic events involved in the full transformation of B cells.

PURPOSE: To describe the features of carcinoembryonic antigen (CEA) that are important for its use in vaccination approaches and review the clinical experience with therapeutic vaccines targeting CEA.METHODS: A PubMed search was performed on CEA, along with various qualifiers such as cancer vaccines, epitopes, and function. Relevant articles were reviewed.RESULTS: CEA is a member of the immunoglobulin supergene family and may play a role in tumorigenesis. CEA protein is processed and presented on major histocompatibility complex (MHC) proteins for multiple alleles, including HLA A2, A3, and A24. T lymphocytes from healthy volunteers and cancer patients can recognize the processed epitopes of CEA and can become activated to lyse CEA-expressing tumors. Therapeutic vaccination approaches that have targeted CEA include vaccination with recombinant CEA protein, CEA anti-idiotype antibodies, and dendritic cells pulsed with agonist epitopes of CEA. Humoral responses have predominantly been induced with the first two approaches, whereas CD4 and CD8 responses, disease stabilization, and even objective clinical responses have been seen with the dendritic cell approach. Recently, CEA-poxvirus vectors encoding CEA and costimulatory molecules such as B7.1 have been shown to be safe and to induce increases in the frequency of T-cell precursors that recognize processed epitopes of CEA presented on MHC class 1 molecules. Disease stabilization has been seen in up to 37% of patients treated with these vaccines.CONCLUSION: Tolerance to CEA in patients with cancer can be overcome with several different vaccination approaches, and such vaccinations are safe and immunologically active. Poxvirus-based vaccines can reproducibly generate T-cell responses to CEA and to tumors expressing CEA. Clinical activity has been seen with poxvirus or dendritic cell approaches. Other approaches are also being explored.

To counteract tumor-induced immune suppression, cancer vaccines are increasingly being combined with immune modulators that can not only reverse immune suppression but also enhance vaccine induced immune responses. We found that metronomic cyclophosphamide (50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients in a phase I clinical study. We emulated these results using transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation. In several different models, mice were given mCPA on alternating weeks (20 mg/kg/day PO) in combination with a DPX vaccine containing relevant peptide antigens every three weeks. In these models, only the combination provided effective and significant long-term control of tumor growth. Notably, we found that the efficacy of the combination was comparable when vaccine was given at the beginning or end of a cycle of mCPA. Metronomic CPA had a pronounced lymphodepletive effect on the vaccine draining lymph node, yet did not reduce the development of antigen-specific CD8+ T cells induced by vaccination as detected by MHC-multimer flow cytometry. Combination treatment also increased cytotoxic T cell activity in the spleen measured by IFN-γ ELISPOT and in vivo cytotoxic T cell assay. Analysis of immune gene signatures in the tumor microenvironment by RT-qPCR detected elevated levels of cytotoxic markers, such as IFN-γ and granzyme B, as well as co-inhibitory markers, such as PD-1 and CTLA-4, in mice treated with combination therapy, the latter providing a strong rational for modulating these pathways with recently available monoclonal antibodies. Analysis of spleen cell populations by flow cytometry indicated that mCPA induced transient lymphodepletion that was marked by a selective expansion of myeloid-derived suppressor cells in the absence of vaccination. Selective depletion of regulatory T cells was not observed, in contrast to other regimens of low dose CPA. These results demonstrate that mCPA provides a complex form of immune modulation that is most effective when combined with active immunization. Using these models we can evaluate other immune modulator agents that may enhance vaccine activity, including checkpoint inhibitors or other immune based therapies. Citation Format: Genevieve Weir, Olga Hrytsenko, Marianne M. Stanford, Neil L. Berinstein, Mohan Karkada, Robert S. Liwski, Marc Mansour. Metronomic cyclophosphamide enhances the immunogenicity and anti-tumor activity of a DepoVax based vaccine and may be further enhanced with inhibitors of CTLA-4 or PD-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5028. doi:10.1158/1538-7445.AM2014-5028

5555 Background: Survivin plays a key role in apoptosis, proliferation and angiogenesis. It is overexpressed in 90% of epithelial OC making it an attractive target. Increases in expression have been correlated with progression and drug resistance. DPX-Survivac is a vaccine containing a mix of survivin HLA class I peptides designed to evoke a cytotoxic T cell response against survivin. This Phase I/Ib de-escalation study reports the safety and immune potency of DPX-Survivac in combination with metronomic low dose oral cyclophosphamide (CPA) in OC. Methods: 30 Stage IIc-IV advanced or recurrent OC patients (pts) with no evidence of disease progression post chemotherapy were enrolled. Dose limiting toxicities (DLT) were defined as ≥gd3 AEs, ≥gd2 allergic or autoimmune reactions by CTCAE v4.03. Immune correlates (MDSCs; T regs; B cells) and vaccine induced T cell immunity (ELISpot; tetramer analysis; multi-parametric intracellular cytokine staining) were assessed in purified PBMC and blood. Clinical response ...

Rituximab is a chimeric anti-CD20 monoclonal antibody that has approval for single agent therapy in the treatment of relapsed/refractory low grade or follicular non-Hodgkin's Lymphoma. In published phase II trials, molecular remissions of PCR detectable t(14; 18) disease in the peripheral blood have been reported in up to 62% of patients by three months. We report a case of a patient who achieved prolonged clinical and molecular remission following a single four week course of Rituximab that has exceeded any previous remission achieved with cheino-radiotherapy. The implications of molecular remission as a surrogate of clinical remission and molecular relapse as a harbinger of clinical relapse are reviewed and discussed.

DPX-Survivac is a peptide vaccine targeting the broadly expressed tumor antigen survivin. The vaccine contains a mixture of survivin HLA class I peptides (A1, A2, A3, A24 and B7) formulated in the novel adjuvanting vaccine platform DepoVaxTM. A phase I clinical study performed in advanced ovarian cancer patients (n=19) demonstrated that the combination of metronomic cyclophosphamide (mCPA) and DPX-Survivac exhibited enhanced immune responses by IFN-γ ELISPOT (p Citation Format: Genevieve M. Weir, Neil L. Berinstein, Olga Hrytsenko, Mohan Karkada, Marianne M. Stanford, Marc Mansour. Metronomic cyclophosphamide, when combined with vaccination, increases the immunogenicity of the vaccine and modulates the tumor microenvironment to provide better anti-tumor activity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A84.

3030 Background: Survivin, a protein involved in regulation of apoptosis, is highly expressed in many tumor types and has reported prognostic value. DPX-Survivac is a cocktail of survivin HLA class I peptides (A1, A2, A3, A24 and B7) formulated in the novel adjuvanting vaccine platform DepoVax. A phase I study examined the safety and immune potency of DPX-Survivac in combination with cyclophosphamide in ovarian cancer patients. Methods: 18 of 19 advanced ovarian cancer patients treated with platinum chemotherapy and showing no disease progression completed their vaccine therapy. Cohort A (6 pts) received three 0.5 mL vaccine injections 3 weeks apart; cohorts B and C (6 pts each) received three 0.1 mL or 0.5 mL vaccine injections in combination with metronomic low dose oral cyclophosphamide. Adverse events were assessed using CTCAE v4.0. Blood was collected to study immune function (MDSCs, T regs and B cells) and vaccine-induced T cell immunity (ELISpot, tetramer analysis and multi-parametric intracellular cytokine staining). Repeated measures of immunity at baseline and after 1, 2 and 3 injections were analyzed using a general linear model. Results: DPX-Survivac was well tolerated with no significant systemic AEs. Local injection AEs occured in all patients. Grade 3 local reactions occured in 3 patients (1 pt in B and 2 pts in C). 11 of 12 patients receiving the combination therapy produced immune responses by at least 2 assays, generally established with one or two vaccinations and increased or maintained with boosters. A dose response was observed, with cohort C patients producing significantly higher magnitude responses (C vs B, P=.013). Low dose cyclophosphamide significantly enhanced the 0.5 ml dose (C vs A, P=.015). Notably, antigen specific CD8 T cells were detected ex vivoi n PBL’s using tetramers and further characterized as polyfunctional by multi-parametric ICS, suggesting a robust immune response. Conclusions: DPX-Survivac is well tolerated and immunogenic. Immune modulation with low dose oral cyclophosphamide can dramatically enhance the immunogenicity of this vaccine. The efficacy of the proposed DPX-Survivac vaccine therapy needs to be tested in a randomized phase II study. Clinical trial information: NCT01416038.