PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

PD-1 combination therapy with IL-2 modifies CD8+ T cell exhaustion program.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Immunohistochemical expression of HER2 in breast cancer: socioeconomic impact of inaccurate tests.

BACKGROUND: Treatment for patients with breast cancer (BC) is guided by human epidermal growth factor receptor 2 (HER2) status. The patient's HER2 status is assessed using US Food and Drug Administration-approved in vitro diagnostic (IVD) immunohistochemical (IHC) tests and laboratory-developed IVD tests. We analysed HER2 testing accuracy using data from the Nordic Immunohistochemistry Quality Control (NordiQC) HER2 IHC programme; results were used in an economic BC treatment model. METHODS: Data were obtained from NordiQC HER2 BC surveys performed from 2008 to 2012. False-negative (FN) and false-positive (FP) rates for approved and laboratory-developed IVDs were used to estimate direct costs, loss of survival, productivity benefit and quality-adjusted life-years. In the absence of consistent and accessible clinical and economic data from countries participating in the NordiQC programme, United States productivity data, healthcare costs and patient numbers were used as a surrogate in order to estimate the potential impact of selecting an approved or laboratory-developed IVDs. RESULTS: In total, 1703 tests were performed. Pooled FN rates were 11% for approved IVDs and 25% for laboratory-developed IVDs; FP rates were 0% and 5%, respectively. Using these FP and FN rates in the economic model and applying them to the United States BC population, approved IVD tests would result in better clinical outcomes, i.e., better survival and fewer disease recurrences/progressions, and lower costs, i.e., total direct costs and lost productivity, versus laboratory-developed IVD tests. Every $1 saved by laboratories by using cheaper reagents could potentially result in approximately $6 additional costs to the healthcare system. CONCLUSIONS: The results of this analysis suggest that incorrect HER2 test results have far-reaching clinical and economic consequences.

A Composite Decision Rule of CD8+ T-cell Density in Tumor Biopsies Predicts Efficacy in Early-stage, Immunotherapy Trials.

PURPOSE: To examine whether CD8+ T-cell numbers in paired tumor biopsies in early-stage clinical trials can be used as an early indicator of clinical benefit for cancer immunotherapies. EXPERIMENTAL DESIGN: Paraffin sections of tumor biopsies were stained immunohistochemically for CD8+ T cells, which were digitally enumerated. The tumor biopsies were from cancer patients in early-phase trials testing novel immunotherapeutic agents. Paired biopsies taken before the start of treatment and on-treatment were compared. A total of 155 patients were used as the training set and an additional 221 patients were used as the validation set. RESULTS: Using the Cox proportional hazard model, a ≥0.9- increase in fold change (FC) on a ln scale in CD8+ T cells (corresponding to a 2.5-fold increase on the linear scale), from baseline, demonstrated a greater association with prolonged progression-free survival and allowed improved differentiation between groups above and below the threshold. Similarly, a ≥6.2 threshold in geometric mean of the on-treatment density (OTD) of T cells, which approximately corresponds to 500 cells/mm2, correlated with longer PFS. The combination of both criteria (FC and OTD) provided the best discrimination between clinically nonactive and active compounds. CONCLUSIONS: We propose that a composite score of CD8+ T-cell density in paired biopsies taken before and on-treatment may be a new biomarker to inform on clinical outcomes in early immunotherapy clinical trials.

Investigating the complex interplay between fibroblast activation protein α-positive cancer associated fibroblasts and the tumor microenvironment in the context of cancer immunotherapy.

Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.

Comprehensive analysis of fibroblast activation protein expression across 23 tumor indications: insights for biomarker development in cancer immunotherapies.

Introduction: Fibroblast activation protein (FAP) is predominantly upregulated in various tumor microenvironments and scarcely expressed in normal tissues. Methods: We analyzed FAP across 1216 tissue samples covering 23 tumor types and 70 subtypes. Results: Elevated FAP levels were notable in breast, pancreatic, esophageal, and lung cancers. Using immunohistochemistry and RNAseq, a correlation between FAP gene and protein expression was found. Evaluating FAP's clinical significance, we assessed 29 cohorts from 12 clinical trials, including both mono and combination therapies with the PD-L1 inhibitor atezolizumab and chemotherapy. A trend links higher FAP expression to poorer prognosis, particularly in RCC, across both treatment arms. However, four cohorts showed improved survival with high FAP, while in four others, FAP had no apparent survival impact. Conclusions: Our results emphasize FAP's multifaceted role in therapy response, suggesting its potential as a cancer immunotherapy biomarker.

Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity from a Phase I Study of Simlukafusp Alfa (FAP-IL2v) in Advanced/Metastatic Solid Tumors.

PURPOSE: Simlukafusp alfa [fibroblast activation protein α-targeted IL2 variant (FAP-IL2v)], a tumor-targeted immunocytokine, comprising an IL2 variant moiety with abolished CD25 binding fused to human IgG1, is directed against fibroblast activation protein α. This phase I, open-label, multicenter, dose-escalation, and extension study (NCT02627274) evaluated the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of FAP-IL2v in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Participants received FAP-IL2v intravenously once weekly. Dose escalation started at 5 mg; flat dosing (≤25 mg) and intraparticipant uptitration regimens (15/20, 20/25, 20/20/35, and 20/35/35 mg) were evaluated. Primary objectives were dose-limiting toxicities, maximum tolerated dose, recommended expansion dose, and pharmacokinetics. RESULTS: Sixty-one participants were enrolled. Dose-limiting toxicities included fatigue (flat dose 20 mg: n = 1), asthenia (25 mg: n = 1), drug-induced liver injury (uptitration regimen 20/25 mg: n = 1), transaminase increase (20/25 mg: n = 1), and pneumonia (20/35/35 mg: n = 1). The uptitration regimen 15/20 mg was determined as the maximum tolerated dose and was selected as the recommended expansion dose. Increases in peripheral blood absolute immune cell counts were seen for all tested doses [NK cells, 13-fold; CD4+ T cells (including regulatory T cells), 2-fold; CD8+ T cells, 3.5-fold] but without any percentage change in regulatory T cells. Clinical activity was observed from 5 mg [objective response rate, 5.1% (n = 3); disease control rate, 27.1% (n = 16)]. Responses were durable [n = 3, 2.8 (censored), 6.3, and 43.4 months]. CONCLUSIONS: FAP-IL2v had a manageable safety profile and showed initial signs of antitumor activity in advanced/metastatic solid tumors.

Phase II Study to Determine the Antitumor Activity and Safety of Simlukafusp Alfa (FAP-IL2v) Combined with Atezolizumab in Esophageal Cancer.

PURPOSE: In this study, we report the results from the esophageal squamous cell carcinoma (SCC) cohort of a phase II, noncomparative, basket study evaluating the antitumor activity and safety of fibroblast activation protein-IL2 variant (FAP-IL2v) plus atezolizumab in patients with advanced/metastatic solid tumors (NCT03386721). PATIENTS AND METHODS: Eligible patients had an Eastern Cooperative Oncology Group performance status of 0 to 1; measurable metastatic, persistent, or recurrent esophageal SCC; progression on ≥1 prior therapy; and were checkpoint inhibitor-naïve. Patients received FAP-IL2v 10 mg plus atezolizumab 1,200 mg intravenously every 3 weeks, or FAP-IL2v weekly for 4 weeks and then every 2 weeks plus atezolizumab 840 mg intravenously every 2 weeks. The primary endpoint was investigator-assessed objective response rate (ORR). RESULTS: In the response-evaluable population (N = 34), the best confirmed ORR was 20.6% [95% confidence interval (CI), 10.4-36.8], with a complete response seen in 1 patient and partial responses in 6 patients. The disease control rate was 44.1% (complete response = 2.9%; partial response = 17.6%; stable disease = 23.5%), and the median duration of response was 10.1 mon/ths (95% CI, 5.6-26.7). The median progression-free survival was 1.9 months (95% CI, 1.8-3.7). Analysis of response by PDL1 expression (Ventana SP263) resulted in an ORR of 26.7% for patients with PDL1-positive tumors (tumor area positivity cutoff ≥1%; n = 15) and 7.1% for patients with PDL1-negative tumors (tumor area positivity cutoff <1%; n = 14). Overall, the treatment combination was tolerable, and adverse events were consistent with the known safety profiles of each drug. CONCLUSIONS: FAP-IL2v plus atezolizumab demonstrated clinical activity and was tolerable in patients with previously treated esophageal SCC.

Phase 1b Study of the Immunocytokine Simlukafusp alfa (FAP-IL2v), in Combination with Cetuximab in Patients with Head and Neck Squamous Cell Carcinoma.

PURPOSE: This phase 1b trial evaluated FAP-IL2v, a novel immune-cytokine engineered to minimize CD25-mediated toxicities, in combination with cetuximab, in patients with recurrent, unresectable, or metastatic head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: Patients received FAP-IL2v either on a continuous weekly (QW) schedule, or QW for 4 weeks and then every 2 weeks (Q2W). Cetuximab was dosed at QW or Q2W schedules. The primary objectives were to evaluate the safety and tolerability, maximum tolerated dose (MTD), pharmacokinetics, and clinical activity for the combination of FAP-IL2v with cetuximab. Exploratory objectives included pharmacodynamic analyses. RESULTS: A total of 58 patients were enrolled, 19 patients into the dose-escalation, and 39 patients into the expansion part. The MTD of FAP-IL2v was defined as 10 mg (QW/Q2W) in combination with cetuximab (500 mg/m2, Q2W), which was further tested in the expansion part. The most common FAP-IL2v-related adverse events with a grade 3 or 4 severity were hypophosphatemia (19%), lymphopenia (16%), and infusion-related reaction (14%). The pharmacokinetics of FAP-IL2v in combination with cetuximab was similar to that after administration as monotherapy. Consistent with the proposed mode-of-action, FAP-IL2v preferentially expanded intratumoral NK and CD8 T cells. Four patients achieved a partial response, the objective response rate was 7% (95% CI: 3.2, 14.7). CONCLUSIONS: The safety profile of FAP-IL2v in combination with cetuximab was acceptable and pharmacodynamic markers support the proposed mode-of-action of this combination, but the overall low antitumor activity does not warrant further clinical exploration in HNSCC. [Part C of Study BP29842 (NCT02627274).].

Phase 2 study of the antitumour activity and safety of simlukafusp alfa (FAP-IL2v) combined with atezolizumab in patients with recurrent and/or metastatic cervical squamous cell carcinoma.

BACKGROUND: Simlukafusp alfa (FAP-IL2v) is an immune cytokine engineered to selectively promote immune responses in the tumour microenvironment. We evaluated the antitumour activity and safety of FAP-IL2v plus atezolizumab in recurrent and/or metastatic cervical squamous cell carcinoma (SCC) in a phase 2 basket study (NCT03386721). METHODS: Patients with confirmed metastatic, persistent or recurrent cervical SCC who had progressed on ≥1 anti-cancer therapy and had measurable disease were enrolled. FAP-IL2v 10 mg was administered once every 3 weeks (Q3W) or once weekly (QW) for 4 weeks then once every 2 weeks (Q2W) with the corresponding Q3W or Q2W atezolizumab regimens. The primary endpoint was objective response rate by investigator assessment. FINDINGS: Forty-eight patients were enrolled (Q3W: n = 47; QW/Q2W: n = 1). Among 45 response evaluable patients, objective responses occurred in 12 patients (27%; CI 16.0-41.0), including 3 complete and 9 partial responses. Responses occurred in 6/19 PD-L1 positive patients (32%; 95% CI 15.4-54.0) and 5/24 PD-L1 negative patients (21%; 95% CI 9.2-35.6). Median duration of response was 13.3 months (95% CI 7.6-NE). Median progression-free survival was 3.7 months (95% CI 3.3-9.0). Adverse events (AEs) were consistent with the known safety profile of each drug. AEs leading to withdrawal of either agent occurred in 6 patients (13%). Pronounced expansion and activation of natural killer and CD8 T cells in peripheral blood and increased tumour infiltration and inflammation were observed. INTERPRETATION: FAP-IL2v plus atezolizumab is clinically active and has manageable safety in patients with recurrent and/or metastatic cervical SCC. FUNDING: F. Hoffmann-La Roche Ltd.

Obinutuzumab Pretreatment as a Novel Approach to Mitigate Formation of Anti-Drug Antibodies Against Cergutuzumab Amunaleukin in Patients with Solid Tumors.

PURPOSE: The immunocytokine cergutuzumab amunaleukin (CEA-IL2v) showed manageable safety and favorable pharmacodynamics in phase I/Ib trials in patients with advanced/metastatic carcinoembryonic antigen-positive (CEA+) solid tumors, but this was accompanied by a high incidence of anti-drug antibodies (ADA). We examined B-cell depletion with obinutuzumab as a potential mitigation strategy. EXPERIMENTAL DESIGN: Preclinical data comparing B-cell depletion with rituximab versus obinutuzumab are summarized. Substudies of phase I/Ib trials investigated the effect of obinutuzumab pretreatment on ADA development, safety, pharmacodynamics, and antitumor activity of CEA-IL2v ± atezolizumab in patients with advanced/metastatic or unresectable CEA+ solid tumors who had progressed on standard of care. RESULTS: Preclinical data showed superior B-cell depletion with obinutuzumab versus rituximab. In clinical studies, patients received CEA-IL2v monotherapy with (n = 16) or without (n = 6) obinutuzumab pretreatment (monotherapy study), or CEA-IL2v + atezolizumab + obinutuzumab pretreatment (n = 5; combination study). In the monotherapy study, after four cycles (every 2 weeks treatment), 0/15 evaluable patients administered obinutuzumab pretreatment had ADAs versus 4/6 patients without obinutuzumab. Obinutuzumab pretreatment with CEA-IL2v monotherapy showed no new safety signals and pharmacodynamic data suggested minimal impact on T cells and natural killer cells. Conversely, increased liver toxicity was observed in the combination study (CEA-IL2v + atezolizumab + obinutuzumab pretreatment). CONCLUSIONS: These preliminary findings suggest that obinutuzumab pretreatment before CEA-IL2v administration in patients with CEA+ solid tumors may be a feasible and potent ADA mitigation strategy, with an acceptable safety profile, supporting broader investigation of obinutuzumab pretreatment for ADA mitigation in other settings.

Virus-specific CD8+ T cells upregulate programmed death-1 expression during acute friend retrovirus infection but are highly cytotoxic and control virus replication.

It was recently reported that inhibitory molecules such as programmed death-1 (PD-1) were upregulated on CD8(+) T cells during acute Friend retrovirus infection and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8(+) T cells upregulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1(hi) and PD-1(lo) subsets. More PD-1(lo) cells produced antiviral cytokines such as IFN-γ and TNF-α, whereas more PD-1(hi) cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8(+) T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus, direct ex vivo analyses and in vivo experiments revealed high CD8(+) T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Prediction of the Optimal Dosing Regimen Using a Mathematical Model of Tumor Uptake for Immunocytokine-Based Cancer Immunotherapy.

Purpose: Optimal dosing is critical for immunocytokine-based cancer immunotherapy to maximize efficacy and minimize toxicity. Cergutuzumab amunaleukin (CEA-IL2v) is a novel CEA-targeted immunocytokine. We set out to develop a mathematical model to predict intratumoral CEA-IL2v concentrations following various systemic dosing intensities.Experimental Design: Sequential measurements of CEA-IL2v plasma concentrations in 74 patients with solid tumors were applied in a series of differential equations to devise a model that also incorporates the peripheral concentrations of IL2 receptor-positive cell populations (i.e., CD8+, CD4+, NK, and B cells), which affect tumor bioavailability of CEA-IL2v. Imaging data from a subset of 14 patients were subsequently utilized to additionally predict antibody uptake in tumor tissues.Results: We created a pharmacokinetic/pharmacodynamic mathematical model that incorporates the expansion of IL2R-positive target cells at multiple dose levels and different schedules of CEA-IL2v. Model-based prediction of drug levels correlated with the concentration of IL2R-positive cells in the peripheral blood of patients. The pharmacokinetic model was further refined and extended by adding a model of antibody uptake, which is based on drug dose and the biological properties of the tumor. In silico predictions of our model correlated with imaging data and demonstrated that a dose-dense schedule comprising escalating doses and shortened intervals of drug administration can improve intratumoral drug uptake and overcome consumption of CEA-IL2v by the expanding population of IL2R-positive cells.Conclusions: The model presented here allows simulation of individualized treatment plans for optimal dosing and scheduling of immunocytokines for anticancer immunotherapy. Clin Cancer Res; 24(14); 3325-33. ©2018 AACRSee related commentary by Ruiz-Cerdá et al., p. 3236.

Overexpression of acid sphingomyelinase sensitizes glioma cells to chemotherapy.

Ceramide has been shown by many studies to induce apoptosis. Therefore, upregulation of ceramide is discussed as a novel approach for tumor treatment. However, it is unknown whether overexpression of acid sphingomyelinase releasing ceramide from sphingomyelin sensitizes cells to chemotherapy and, thus, serves as a potential target to amplify chemotherapy. Here, the authors demonstrate that transfection of human or murine glioma cells with acid sphingomyelinase results in a marked sensitization of glioma cells to gemcitabine and doxorubicin, respectively. Transfected cells responded to chemotherapy with an increased activation of acid sphingomyelinase, elevated ceramide levels, and approximately fourfold higher rates of cell death than control transfected cells. Neutralization of reactive oxygen species prevented these events. The data indicate a significant sensitization of glioma cells to chemotherapy treatment by expression of acid sphingomyelinase and further suggest an activation of acid sphingomyelinase by gemcitabine or doxorubicin, respectively, via reactive oxygen species.

Ceramide-enriched membrane domains.

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.

Percutaneous MR-guided radiofrequency ablation of recurrent sacrococcygeal chordomas.

OBJECTIVE: Radiofrequency ablation is emerging as a therapeutic technique for the treatment of an increasing variety of tumors. Exact visual guidance to the tumor and controlled delivery of energy is pivotal for ablation success. CONCLUSION: Introducing MRI as a guidance technique ideally uses tumor-specific tissue characteristics, allows direct multiplanar reconstruction for precise needle positioning, and permits real-time monitoring and assessment of treatment-induced tissue signal changes to increase the safety of the procedure.

Girdin (GIV) Expression as a Prognostic Marker of Recurrence in Mismatch Repair-Proficient Stage II Colon Cancer.

PURPOSE: Prognostic markers that identify patients with stage II colon cancers who are at the risk of recurrence are essential to personalize therapy. We evaluated the potential of GIV/Girdin as a predictor of recurrence risk in such patients. EXPERIMENTAL DESIGN: Expression of full-length GIV was evaluated by IHC using a newly developed mAb together with a mismatch repair (MMR)-specific antibody panel in three stage II colon cancer patient cohorts, that is, a training (n = 192), test (n = 317), and validation (n = 181) cohort, with clinical follow-up data. Recurrence risk stratification models were established in the training cohort of T3, proficient MMR (pMMR) patients without chemotherapy and subsequently validated. RESULTS: For T3 pMMR tumors, GIV expression and the presence of lymphovascular invasion (LVI) were the only factors predicting recurrence in both training (GIV: HR, 2.78, P = 0.013; LVI: HR, 2.54, P = 0.025) and combined test and validation (pooled) cohorts (GIV: HR, 1.85, P = 0.019; LVI: HR, 2.52, P = 0.0004). A risk model based on GIV expression and LVI status classified patients into high- or low-risk groups; 3-year recurrence-free survival was significantly lower in the high-risk versus low-risk group across all cohorts [Training: 52.3% vs. 84.8%; HR, 3.74, 95% confidence interval (CI), 1.50-9.32; Test: 85.9% vs. 97.9%, HR, 7.83, 95% CI, 1.03-59.54; validation: 59.4% vs. 84.4%, HR, 3.71, 95% CI, 1.24-11.12]. CONCLUSIONS: GIV expression status predicts recurrence risk in patients with T3 pMMR stage II colon cancer. A risk model combining GIV expression and LVI status information further enhances prediction of recurrence. Further validation studies are warranted before GIV status can be routinely included in patient management algorithms. Clin Cancer Res; 22(14); 3488-98. ©2016 AACR.

Specific inhibition of fibroblast activation protein (FAP)-alpha prevents tumor progression in vitro.

PURPOSE: Solid tumors modulate their environment to keep non-malignant stromal cells in a tumor-promoting state. The main cells in the stroma of epithelial derived tumors are cancer associated fibroblasts (CAF) that are critical to tumorigenesis and angiogenesis. CAFs also supply the tumor cells with growth factors and extracellular matrix (ECM) degrading enzymes. They are thus essential for tumor initiation as well as tumor progression and metastasis, suggesting that they represent an ideal cellular target of an integrative tumor therapy. Fibroblast activation protein (FAP) is a well-defined marker, expressed at high levels on the cell surface of CAFs. FAP, a constitutively active serine peptidase with both dipeptidyl peptidase IV (DPP IV) and collagenase/gelatinase activity, promotes malignant and invasive behavior of epithelial cancers. High stromal expression levels of FAP correlate with poor prognosis. FAP is difficult to detect in non-diseased adult tissue, but it is generally expressed at sites of tissue remodeling. MATERIALS AND METHODS: In our experiments, we aimed for a reduction of the pro-tumorigenic activities of CAFs by depleting FAP from fibroblasts growing in a composite environment with epithelial tumor cells. RESULTS: FAP depletion was achieved by two therapeutically relevant approaches: a novel internalizing anti-FAP IgG1 antibody and FAP gene knock-down by siRNA delivery. The antibody effectively removed FAP from the cell surface and was capable of reversing the FAP mediated migratory and invasive capacity. FAP RNA interference was equally effective when compared to the antibody. CONCLUSIONS: Thus, targeting FAP on CAF suppresses pro-tumorigenic activities and may result in a clinically effective reduction of tumor progression and dissemination.

5-Fluorouracil and interferon-α immunochemotherapy enhances immunogenicity of murine pancreatic cancer through upregulation of NKG2D ligands and MHC class I.

Pancreatic cancer has the poorest prognosis of all gastrointestinal cancers, driving the need for new therapeutic approaches. Adjuvant 5-fluorouacil (5-FU) chemotherapy proved effective in increasing the survival of patients with resected tumors. Furthermore, the addition of interferon alpha (IFN-α) immunotherapy to 5-FU has shown encouraging clinical results. The aim of this study was to determine the relevance of different immune cell populations, namely natural killer (NK) cells, CD8 T cells, and dendritic cells, in the anticancer immune response mediated by the combination therapy using an orthotopic mouse model of pancreatic carcinoma and to get more insight into the underlying mechanisms of action. Depleting CD8 T cells, NK cells, or dendritic cells significantly reduced the anticancer effects mediated by the combination therapy. Tumors of mice treated with 5-FU+IFN-α harbored higher numbers of infiltrating NK cells in comparison with control mice. In addition, NK cells isolated from these mice showed enhanced cytotoxicity against Panc02 pancreatic cancer cells. Furthermore, 5-FU+IFN-α treatment increased the expression of major histocompatibility complex class I and NKG2D ligands on Panc02 cells that could be a potential key for enhancing the immunogenicity of tumors. Understanding how this combination therapy enhances the immunogenicity of pancreatic tumors in our model may provide potential predictive biomarkers. This will allow to evaluate the efficacy of this immunochemotherapy more effectively in future clinical trials and to identify patients who will benefit most from it.