Modelling the lymphatic metastatic progression pathways of OPSCC from multi-institutional datasets.

The elective clinical target volume (CTV-N) in oropharyngeal squamous cell carcinoma (OPSCC) is currently based mostly on the prevalence of lymph node metastases in different lymph node levels (LNLs) for a given primary tumor location. We present a probabilistic model for ipsilateral lymphatic spread that can quantify the microscopic nodal involvement risk based on an individual patient's T-category and clinical involvement of LNLs at diagnosis. We extend a previously published hidden Markov model (HMM), which models the LNLs (I, II, III, IV, V, and VII) as hidden binary random variables (RVs). Each represents a patient's true state of lymphatic involvement. Clinical involvement at diagnosis represents the observed binary RVs linked to the true state via sensitivity and specificity. The primary tumor and the hidden RVs are connected in a graph. Each edge represents the conditional probability of metastatic spread per abstract time-step, given disease at the edge's starting node. To learn these probabilities, we draw Markov chain Monte Carlo samples from the likelihood of a dataset (686 OPSCC patients) from three institutions. We compute the model evidence using thermodynamic integration for different graphs to determine which describes the data best.The graph maximizing the model evidence connects the tumor to each LNL and the LNLs I through V in order. It predicts the risk of occult disease in level IV is below 5% if level III is clinically negative, and that the risk of occult disease in level V is below 5% except for advanced T-category (T3 and T4) patients with clinical involvement of levels II, III, and IV. The provided statistical model of nodal involvement in OPSCC patients trained on multi-institutional data may guide the design of clinical trials on volume-deescalated treatment of OPSCC and contribute to more personal guidelines on elective nodal treatment.

Toward a global harmonization of service infrastructure in academic clinical trial units: an international survey.

Background: Clinicians around the world perform clinical research in addition to their high workload. To meet the demands of high quality Investigator Initiated Trials (IITs), Clinical Trial Units (CTUs) (as part of Academic Research Institutions) are implemented worldwide. CTUs increasingly hold a key position in facilitating the international mutual acceptance of clinical research data by promoting clinical research practices and infrastructure according to international standards. Aim: In this project, we aimed to identify services that established and internationally operating CTUs - members of the International Clinical Trial Center Network (ICN) - consider most important to ensure the smooth processing of a clinical trial while meeting international standards. We thereby aim to drive international harmonization by providing emerging and growing CTUs with a resource for informed service range set-up. Methods: Following the AMEE Guide, we developed a questionnaire, addressing the perceived importance of different CTU services. Survey participants were senior representatives of CTUs and part of the ICN with long-term experience in their field and institution. Results: Services concerning quality and coordination of a research project were considered to be most essential, i.e., Quality management, Monitoring and Project management, followed by Regulatory & Legal affairs, Education & Training, and Data management. Operative services for conducting a research project, i.e., Study Nurse with patient contact and Study Nurse without patient contact, were considered to be least important. Conclusion: To balance the range of services offered while meeting high international standards of clinical research, emerging CTUs should focus on offering (quality) management services and expertise in regulatory and legal affairs. Additionally, education and training services are required to ensure clinicians are well trained on GCP and legislation. CTUs should evaluate whether the expertise and resources are available to offer operative services.

The carbon footprint of clinical trials: a global survey on the status quo and current regulatory guidance.

INTRODUCTION: All economic sectors including the service sector, along with healthcare, education and research, need to reduce their greenhouse gas emissions to limit global temperature increases. In this study, we aim to globally assess the awareness and current actions taken by Academic Research Institutions (ARIs) or governments regarding the reduction of carbon dioxide equivalent (CO2e) emissions for clinical research. METHODS: We designed a cross-sectional survey-based study, which was distributed within the International Clinical Trials Center Network (ICN). The survey population comprised representatives of the ICN who had extensive experience in academic clinical research and profound knowledge and understanding of the local context. RESULTS: The response rate was 80%. Responding ARIs were from 15 different countries and 4 continents. Around half of the ARIs reported that almost none of their research projects considered reducing their carbon footprint. The other half of the ARIs were not familiar with this subject at all. According to 60% of the respondents, greenhouse gas emissions are not assessed by Institutional Review Boards (IRBs)/Ethics Committees (ECs) or competent authorities, while 40% did not know. Neither IRBs/ECs nor competent authorities currently advise sponsors and investigators on reducing the carbon footprint of their clinical research projects. As for reducing greenhouse gas emissions in clinical research, virtual conferences and meetings were the most commonly implemented measures by ARIs across all regions. Finally, we have put together an action plan/checklist advising researchers on carbon footprint reduction for clinical trials. CONCLUSION: Currently, greenhouse gas emissions are neglected during the planning phase of a research project, and they are not yet addressed or assessed by default during the approval procedures by IRBs/ECs or competent authorities. Thus, all involved stakeholders within clinical research need to be made aware of it through advice from ARIs and IRBs/ECs, among others.

A dataset on patient-individual lymph node involvement in oropharyngeal squamous cell carcinoma.

Dataset: We provide a dataset on lymph node level (LNL) involvement in 287 patients with newly diagnosed oropharyngeal squamous cell carcinoma (OPSCC). For each patient, ipsilateral and contralateral LNL involvement for levels I to VII is reported together with clinicopathological factors including TNM-stage, primary tumor subsite, tumor lateralization, HPV status, sex, age, smoking status, and primary treatment. LNL involvement was assessed individually based on available diagnostic modalities (PET, MRI, CT, fine needle aspiration) by reviewing pathology and radiology reports together with the radiological images. The data is shared as a CSV-table with rows of patients and columns of patient/tumor-specific information and the involvement of individual LNL based on the respective diagnostic modalities. Reuse potential: Patterns of lymphatic progression have never been reported on a patient-individual basis in as much detail as provided in this dataset. The data can be used to build quantitative models for lymphatic tumor progression to estimate the probability of occult metastases in LNLs. This may in turn allow for further personalization of the elective clinical target volume definition in radiotherapy and the extent of neck dissection for surgically treated patients. The data can be pooled with other data to build large multi-institutional datasets on lymphatic metastatic progression in the future. Co-submission: This paper supports the original scientific article by Roman Ludwig, Jean-Marc Hoffmann, Bertrand Pouymayou, Grégoire Morand, Martina Broglie Däppen, Matthias Guckenberger, Vincent Grégoire, Panagiotis Balermpas, Jan Unkelbach, "Detailed patient-individual reporting of lymph node involvement in oropharyngeal squamous cell carcinoma with an online interface", Radiotherapy & Oncology [1].

Detailed patient-individual reporting of lymph node involvement in oropharyngeal squamous cell carcinoma with an online interface.

PURPOSE/OBJECTIVE: Whereas the prevalence of lymph node level (LNL) involvement in head & neck squamous cell carcinomas (HNSCC) has been reported, the details of lymphatic progression patterns are insufficiently quantified. In this study, we investigate how the risk of metastases in each LNL depends on the involvement of upstream LNLs, T-category, HPV status and other risk factors. MATERIALS/METHODS: We retrospectively analyzed patients with newly diagnosed oropharyngeal squamous cell carcinoma (OPSCC) treated at a single institution, resulting in a dataset of 287 patients. For all patients, involvement of LNLs I-VII was recorded individually based on available diagnostic modalities (PET, MRI, CT, FNA) together with clinicopathological factors. To analyze the dataset, a web-based graphical user interface (GUI) was developed, which allows querying the number of patients with a certain combination of co-involved LNLs and tumor characteristics. RESULTS: The full dataset and GUI is part of the publication. Selected findings are: Ipsilateral level IV was involved in 27% of patients with level II and III involvement, but only in 2% of patients with level II but not III involvement. Prevalence of involvement of ipsilateral levels II, III, IV, V was 79%, 34%, 7%, 3% for early T-category patients (T1/T2) and 85%, 50%, 17%, 9% for late T-category (T3/T4), quantifying increasing involvement with T-category. Contralateral levels II, III, IV were involved in 41%, 19%, 4% and 12%, 3%, 2% for tumors with and without midline extension, respectively. T-stage dependence of LNL involvement was more pronounced in HPV negative than positive tumors, but overall involvement was similar. Ipsilateral level VII was involved in 14% and 6% of patients with primary tumors in the tonsil and the base of tongue, respectively. CONCLUSIONS: Detailed quantification of LNL involvement in HNSCC depending on involvement of upstream LNLs and clinicopathological factors may allow for further personalization of CTV-N definition in the future.

Comparison of IL-2 vs IL-7/IL-15 for the generation of NY-ESO-1-specific T cells.

The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naïve-like T cell (TN) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4+ T cells and a decrease of CD8+ T cells, enriched the amount of TN among CD4+ T cells but not among CD8+ T cells. In a 51Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of TN. However, the absolute number of TN did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems.

Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies.

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance.

Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients.

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.

Modulation of B Cells and Homing Marker on NK Cells Through Extracorporeal Photopheresis in Patients With Steroid-Refractory/Resistant Graft-Vs.-Host Disease Without Hampering Anti-viral/Anti-leukemic Effects.

Graft-vs.-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation, significantly affects the post-transplant morbidity and mortality. Systemic steroids remain the gold standard for the initial management of GvHD. However, up to 60% of patients will not sufficiently respond to steroids. Extracorporeal photopheresis (ECP), a cell-based immunotherapy, has shown good clinical results in such steroid-refractory/resistant GvHD patients. Given its immunomodulatory, but not global immunosuppressive and steroid-sparing capacity, ECP constitutes an attractive option. In the case of GvHD, the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells. ECP therapy may restore this balance. However, the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear. In this study, 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD). A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining, LuminexTM-based cytokine, interferon-γ enzyme-linked immunospot, and chromium-51 release assays. Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis. Immunologically, modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L+ NK cells and newly defined CD19hiCD20hi B cells were modulated, but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved. (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33-CD11b+) to an activated subset (CD33+CD11b+). (4) The frequency of Foxp3+CD4+ regulatory T cells (Tregs) and CD24+CD38hi regulatory B cells was considerably increased in aGvHD patients, and Foxp3+CD8+ Tregs in cGvHD patients. (5) Proinflammatory cytokines like IL-1ß, IL-6, IL-8, and TNF-α were significantly reduced. In summary, ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects.

Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells.

Enhanced in vivo expansion, long-term persistence of chimeric antigen receptor T (CART) cells, and efficient tumor eradication through these cells are linked to the proportion of less-differentiated cells in the CART cell product. Retronectin is well established as an adjuvant for improved retroviral transduction, while its property to enrich less-differentiated T cells is less known. In order to increase these subsets, this study investigated the effects of retronectin-mediated T-cell activation for CD19-specific CART cell production. Peripheral blood mononuclear cells of healthy donors and untreated chronic lymphocytic leukemia (CLL) patients without or with positive selection for CD3+ T cells were transduced with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Activation of peripheral blood mononuclear cells was performed by CD3/CD28, CD3/CD28/retronectin, or CD3/retronectin. Interleukin-7 and -15 were supplemented to all cultures. Retronectin was used in all three activation protocols for retroviral transduction. Expansion was assessed by trypan blue staining. Viability, transduction efficiency, immune phenotype, and cytokine production were longitudinally analyzed by flow cytometry. Cytotoxic capacity of generated CART cells was evaluated using a classical chromium-51 release assay. Retronectin-mediated activation resulted in an enrichment of CD8+ cytotoxic CART cells and less-differentiated naïve-like T cells (CD45RA+CCR7+). Retronectin-activated CART cells showed increased cytotoxic activity. However, activation with retronectin decreased viability, expansion, transduction efficiency, and cytokine production, particularly of CLL patient-derived CART cells. Both retronectin-mediated activation protocols promoted a less-differentiated CART cell phenotype without comprising cytotoxic properties of healthy donor-derived CART cells. However, up-front retronectin resulted in reduced viability and expansion in CLL patients. This effect is probably attributed to the retronectin-mediated activation of B cells with prolonged CLL persistence. Consequently, CART cell expansion and generation failed. In summary, activation with retronectin should be performed with caution and may be limited to patients without a higher percentage of tumor cells in the peripheral blood.

Chimeric antigen receptor transduced T cells: Tuning up for the next generation.

Chimeric antigen receptor (CAR) T cell therapy has recently achieved impressive clinical outcome in patients with CD19-positive hematologic malignancies. Extrapolation of CAR T cell treatment to solid tumors, however, has not yet yielded similar results. This might be due to intrinsic causes, e.g. insufficient CAR T cell activation or CAR toxicity as well as extrinsic factors displaying an unfavorable tumor environment for CAR T cells by raising physical and chemical barriers. In this review, we discuss the advantages as well as major obstacles of CAR T cell therapy, particularly in the context of solid tumors, and focus on efforts and novel strategies in CAR T cell development.

Next-generation dendritic cell-based vaccines for leukemia patients.

Up to today treatment of leukemia patients remains challenging and different therapies have been developed, among them the generation of dendritic cell (DC) vaccines. DCs, highly specific for immunogenic cancer antigens, are generated either ex vivo or in vivo and boost the immune response against leukemic cells. Nevertheless, response rates are still heterogeneous and DC vaccines need improvement. New methods for generating DC vaccines have been summed up under the term 'next-generation DC vaccines'. They range from the analysis of human leukocyte antigen-ligandomes to immunogenic cell death inducers, from the production of viral vectors to mRNA transfection and finally from delivering peptides to DCs in vivo through either antibodies or cell-penetrating peptides. This review gives an overview of the latest developments in this still evolving field.

Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients.

INTRODUCTION: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (TN) vs. effector (TE) T cells, TN cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TN/TE ratio of CART cells. MATERIALS AND METHODS: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays. RESULTS: IL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (TSCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (TEM), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTN cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTN cells in untreated CLL patients. Final TN/TE ratio stayed <0.3 despite stimulation condition for patients, whereas this ratio was >2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion. CONCLUSION: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product.

Chimeric Antigen Receptor T Cell Therapy Targeting CD19-Positive Leukemia and Lymphoma in the Context of Stem Cell Transplantation.

Novel therapies with chimeric antigen receptor (CAR)-transduced T cells (TCs) sparked new hope for patients with relapsed or refractory CD19-positive leukemia or lymphoma even after stem cell therapies. This review focuses on CARs recognizing the B cell antigen CD19. Both retroviral and lentiviral vectors are used, encoding various anti-CD19 CAR constructs comprising costimulatory molecules such as CD28, CD137/4-1BB, and OX40 either alone (second-generation CARs) or in combination (third-generation CARs). Current, up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 58 ongoing trials is given. Clinical responses were achieved in up to 81% of ALL, 50% of CLL, and 40% of NHL patients. Factors with potential influence on the clinical outcome might be the design of the vector, the preconditioning regimen, and the number and quality of transfused CAR TCs. The applicability of clinical CAR TC therapy might include relapse after allogeneic stem cell transplantation (alloSCT), and ineligibility for or "bridging" until alloSCT. In summary, CAR therapy represents a highly promising treatment option even in heavily pretreated patients.

Progress of dendritic cell-based cancer vaccines for patients with hematological malignancies.

INTRODUCTION: Dendritic cells (DCs) are the most professional antigen-presenting cells eliciting cellular and humoral immune responses against cancer cells by expressing these antigens on MHC class I/II complexes to T cells. Therefore, they have been employed in many clinical trials as cancer vaccines for patients with cancer. This review focuses on the use of DCs in leukemia patients expressing leukemia-associated antigens (LAAs). AREAS COVERED: The contribution of both stimulating vs. tolerogenic DCs as well as of other factors to the milieu of anti-leukemia immune responses are discussed. Several DC vaccination strategies like leukemia lysate, proteins and peptides have been developed. Next generation DC vaccines comprise transduction of DCs with retroviral vectors encoding for LAAs, cytokines and costimulatory molecules as well as transfection of DCs with naked RNA encoding for LAAs. Published as well as ongoing clinical trials are reported and critically reviewed. EXPERT OPINION: Future results will demonstrate whether next-generation DCs are really superior to conventional pulsing with peptide, protein or tumor lysate. However, currently available methods based on nucleic acid transfection/transduction are tempting in terms of material production costs and time for clinical application according to good manufacturing practice (GMP).