R Wu

Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients.

Purpose: Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) is a promising treatment for metastatic melanoma unresponsive to conventional therapies. We report here on the results of an ongoing phase II clinical trial testing the efficacy of ACT using TIL in patients with metastatic melanoma and the association of specific patient clinical characteristics and the phenotypic attributes of the infused TIL with clinical response. Experimental Design: Altogether, 31 transiently lymphodepleted patients were treated with their expanded TIL, followed by two cycles of high-dose interleukin (IL)-2 therapy. The effects of patient clinical features and the phenotypes of the T cells infused on the clinical response were determined. Results: Overall, 15 of 31 (48.4%) patients had an objective clinical response using immune-related response criteria (irRC) with 2 patients (6.5%) having a complete response. Progression-free survival of more than 12 months was observed for 9 of 15 (60%) of the responding patients. Factors significantly associated with the objective tumor regression included a higher number of TIL infused, a higher proportion of CD8+ T cells in the infusion product, a more differentiated effector phenotype of the CD8+ population, and a higher frequency of CD8+ T cells coexpressing the negative costimulation molecule “B- and T-lymphocyte attenuator” (BTLA). No significant difference in the telomere lengths of TIL between responders and nonresponders was identified. Conclusion: These results indicate that the immunotherapy with expanded autologous TIL is capable of achieving durable clinical responses in patients with metastatic melanoma and that CD8+ T cells in the infused TIL, particularly differentiated effectors cells and cells expressing BTLA, are associated with tumor regression. Clin Cancer Res; 18(24); 6758–70. ©2012 AACR.

Adoptive T-cell Therapy Using Autologous Tumor-infiltrating Lymphocytes for Metastatic Melanoma: Current Status and Future Outlook

Immunotherapy using autologous T cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs), T cells transduced with high-affinity T cell receptors against major tumor antigens, and T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin light chains with endodomains of T-cell signaling molecules. Among these and other options for T-cell therapy, TILs together with high-dose interleukin 2 have had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined and undefined tumors antigens against all possible major histocompatibility complex, rather than the single specificity and limited major histocompatibility complex coverage of the newer T cell receptors and chimeric antigen receptor transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL-mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trial is needed in an attempt to gain regulatory approval of TILs as standard of care. Second, improvements in how we expand TILs for therapy are needed that minimize the time the T cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers to better select suitable patients for TIL therapy to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TILs will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TILs with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation should further increase therapeutic efficacy and durable complete response rates.

Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function.

Adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) with high-dose interleukin-2 is a promising form of immunotherapy for stage IV melanoma having clinical response rates of 50% or more. One of the major problems preventing further success of this therapy is that the current protocols used to highly expand TIL for infusion drive CD8+ T cells to differentiate into effector cells losing key costimulatory molecules such as CD28 and CD27. This has been associated with a lack of persistence in vivo for reasons not entirely clear. In this study, we demonstrate that while human melanoma CD8+ TIL lost CD27 and CD28 expression during the rapid expansion for ACT, they gained expression of the alternative costimulatory molecule CD137/4-1BB, and to a lesser extent CD134/OX40. Postrapid expansion protocol (REP) TIL were found to be highly sensitive to activation-induced cell death when reactivated through the T-cell receptor with low levels of OKT3 antibody. However, coligation of 4-1BB using 2 different agonistic anti-4-1BB antibodies potently prevented activation-induced cell death of post-REP CD8+ TIL, including those specific for melanoma antigen recognized by T cells, and facilitated even further cell expansion. This was correlated with increased levels of bcl-2 and bcl-xL together with decreased bim expression. 4-1BB costimulated post-REP TIL also expressed increased levels of the cytolytic granule proteins and exhibited enhanced cytotoxic T-cell activity against melanoma cells. Lastly, post-REP CD8+ TIL were protected from cell death by anti-4-1BB ligation when exposed to human leukocyte antigen-matched melanoma cells. Our results indicate that 4-1BB costimulation may significantly improve TIL survival during melanoma ACT and boost antitumor cytolytic activity.

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP. We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

Clinical Implementation of Intensity Modulated Proton Therapy for Thoracic Malignancies

PURPOSE Intensity modulated proton therapy (IMPT) can improve dose conformality and better spare normal tissue over passive scattering techniques, but range uncertainties complicate its use, particularly for moving targets. We report our early experience with IMPT for thoracic malignancies in terms of motion analysis and management, plan optimization and robustness, and quality assurance. METHODS AND MATERIALS Thirty-four consecutive patients with lung/mediastinal cancers received IMPT to a median 66 Gy(relative biological equivalence [RBE]). All patients were able to undergo definitive radiation therapy. IMPT was used when the treating physician judged that IMPT conferred a dosimetric advantage; all patients had minimal tumor motion (<5 mm) and underwent individualized tumor-motion dose-uncertainty analysis and 4-dimensional (4D) computed tomographic (CT)-based treatment simulation and motion analysis. Plan robustness was optimized by using a worst-case scenario method. All patients had 4D CT repeated simulation during treatment. RESULTS IMPT produced lower mean lung dose (MLD), lung V5 and V20, heart V40, and esophageal V60 than did IMRT (P<.05) and lower MLD, lung V20, and esophageal V60 than did passive scattering proton therapy (PSPT) (P<.05). D5 to the gross tumor volume and clinical target volume was higher with IMPT than with intensity modulated radiation therapy or PSPT (P<.05). All cases were analyzed for beam-angle-specific motion, water-equivalent thickness, and robustness. Beam angles were chosen to minimize the effect of respiratory motion and avoid previously treated regions, and the maximum deviation from the nominal dose-volume histogram values was kept at <5% for the target dose and met the normal tissue constraints under a worst-case scenario. Patient-specific quality assurance measurements showed that a median 99% (range, 95% to 100%) of the pixels met the 3% dose/3 mm distance criteria for the γ index. Adaptive replanning was used for 9 patients (26.5%). CONCLUSIONS IMPT using 4D CT-based planning, motion management, and optimization was implemented successfully and met our quality assurance parameters for treating challenging thoracic cancers.

A procedure for calculation of monitor units for passively scattered proton radiotherapy beams

The purpose of this study is to validate a monitor unit (MU) calculation procedure for passively scattered proton therapy beams. The output dose per MU (d/MU) of a therapeutic radiation beam is traditionally calibrated under specific reference conditions. These conditions include beam energy, field size, suitable depth in water or water equivalent phantom in a low dose gradient region with known relative depth dose, and source to point of calibration distance. Treatment field settings usually differ from these reference conditions leading to a different d/MU that needs to be determined for delivering the prescribed dose. For passively scattered proton beams, the proton specific parameters, which need to be defined, are related to the energy, lateral scatterers, range modulating wheel, spread out Bragg peak (SOBP) width, thickness of any range shifter, the depth dose value relative to the normalization point in the SOBP, and scatter both from the range compensator and inhomogeneity in the patient. Following the custom for photons or electrons, a set of proton dosimetry factors, representing the changes in the d/MU relative to a reference condition, can be defined as the relative output factor (ROF), SOBP factor (SOBPF), range shifter factor (RSF), SOBP off-center factor (SOBPOCF), off-center ratio (OCR), inverse square factor (ISF), field size factor (FSF), and compensator and patient scatter factor (CPSF). The ROF, SOBPF, and RSF are the major contributors to the d/MU and were measured using an ion chamber in water tank during the clinical commissioning of each beam to create a dosimetry beam data table to be used for calculating the monitor units. The following simple formula is found to provide an independent method to determine the d/MU at the point of interest (POI) in the patient, namely, (d/MU) = ROF SOBPF. RSF SOBPOCF.OCR.FSF.ISF.CPSF. The monitor units for delivering the intended dose (D) to the POI can be obtained from MU = D / (d/MU). The accuracy and robustness of the above formula were validated by calculating the d/MU in water for many different combinations of beam parameters and comparing it with the corresponding measured d/MU by an ion chamber in a water or water/plastic phantom. This procedure has been in use for MU calculation for patient treatment fields at our facility since May 2006. The differences in the calculated and measured values of the d/MU for 623 distinct fields used for patient treatment during the period of May 2006 to February 2007 are within 2% for 99% of these fields. The authors conclude that an intuitive formula similar to the one used for monitor unit calculation of therapeutic photon beams can be used to compute the monitor units of passively scattered proton therapy beams.

Spot-scanning beam proton therapy vs intensity-modulated radiation therapy for ipsilateral head and neck malignancies: a treatment planning comparison.

Radiation therapy for head and neck malignancies can have side effects that impede quality of life. Theoretically, proton therapy can reduce treatment-related morbidity by minimizing the dose to critical normal tissues. We evaluated the feasibility of spot-scanning proton therapy for head and neck malignancies and compared dosimetry between those plans and intensity-modulated radiation therapy (IMRT) plans. Plans from 5 patients who had undergone IMRT for primary tumors of the head and neck were used for planning proton therapy. Both sets of plans were prepared using computed tomography (CT) scans with the goals of achieving 100% of the prescribed dose to the clinical target volume (CTV) and 95% to the planning TV (PTV) while maximizing conformity to the PTV. Dose-volume histograms were generated and compared, as were conformity indexes (CIs) to the PTVs and mean doses to the organs at risk (OARs). Both modalities in all cases achieved 100% of the dose to the CTV and 95% to the PTV. Mean PTV CIs were comparable (0.371 IMRT, 0.374 protons, p = 0.953). Mean doses were significantly lower in the proton plans to the contralateral submandibular (638.7 cGy IMRT, 4.3 cGy protons, p = 0.002) and parotid (533.3 cGy IMRT, 48.5 cGy protons, p = 0.003) glands; oral cavity (1760.4 cGy IMRT, 458.9 cGy protons, p = 0.003); spinal cord (2112.4 cGy IMRT, 249.2 cGy protons, p = 0.002); and brainstem (1553.52 cGy IMRT, 166.2 cGy protons, p = 0.005). Proton plans also produced lower maximum doses to the spinal cord (3692.1 cGy IMRT, 2014.8 cGy protons, p = 0.034) and brainstem (3412.1 cGy IMRT, 1387.6 cGy protons, p = 0.005). Normal tissue V10, V30, and V50 values were also significantly lower in the proton plans. We conclude that spot-scanning proton therapy can significantly reduce the integral dose to head and neck critical structures. Prospective studies are underway to determine if this reduced dose translates to improved quality of life.

PD-1 and BTLA and CD8+ T-cell "exhaustion" in cancer "Exercising" an alternative viewpoint

The elevated expression of PD-1, BTLA, and other co-inhibitory molecules on T cells from cancer patients has become an accepted signature for a state called T-cell “exhaustion” that has emerged almost as dogma in the field. However, here we propose that in some cases this “exhausted” T-cell phenotype may instead be an indicator of T cells that are in a more heightened state of T-cell activation more susceptible to negative regulation rather than being “exhausted.” This alternative interpretation fits in line with the view that CD8+ T-cell activation in cancer results from a continuum of signals regulating their differentiation towards potent effector cells.

Toward a model-based patient selection strategy for proton therapy: External validation of photon-derived normal tissue complication probability models in a head and neck proton therapy cohort

OBJECTIVE To externally validate head and neck cancer (HNC) photon-derived normal tissue complication probability (NTCP) models in patients treated with proton beam therapy (PBT). METHODS This prospective cohort consisted of HNC patients treated with PBT at a single institution. NTCP models were selected based on the availability of data for validation and evaluated by using the leave-one-out cross-validated area under the curve (AUC) for the receiver operating characteristics curve. RESULTS 192 patients were included. The most prevalent tumor site was oropharynx (n=86, 45%), followed by sinonasal (n=28), nasopharyngeal (n=27) or parotid (n=27) tumors. Apart from the prediction of acute mucositis (reduction of AUC of 0.17), the models overall performed well. The validation (PBT) AUC and the published AUC were respectively 0.90 versus 0.88 for feeding tube 6months PBT; 0.70 versus 0.80 for physician-rated dysphagia 6months after PBT; 0.70 versus 0.68 for dry mouth 6months after PBT; and 0.73 versus 0.85 for hypothyroidism 12months after PBT. CONCLUSION Although a drop in NTCP model performance was expected for PBT patients, the models showed robustness and remained valid. Further work is warranted, but these results support the validity of the model-based approach for selecting treatment for patients with HNC.

Chemoradiation therapy using cyclopamine-loaded liquid-lipid nanoparticles and lutetium-177-labeled core-crosslinked polymeric micelles.

Cyclopamine (CPA), a potent inhibitor of the Hedgehog pathway, has produced promising anticancer results in a number of preclinical studies. CPA has also been found to enhance tumor response to radiation therapy. However, CPA is water insoluble. A drug delivery system suitable for systemic administration of CPA is needed before CPA can be considered for clinical translation. We hypothesized that CPA solubilized in a liquid-lipid nanoparticle system (CPA-LLP) for intravenous injection would have desirable pharmacokinetic properties and increased anticancer efficacy. We further hypothesized that CPA-LLP would enhance the response of tumor cells to targeted radiotherapy delivered selectively through intratumoral injection of lutetium-177 bound to core-crosslinked polymeric micelles (CCPM-(177)Lu). We tested the combination therapy in 4T1 murine breast cancer and Miapaca-2 human pancreatic adenocarcinoma models. The results showed that CPA-LLP had higher antitumor cytotoxicity than free CPA (IC50 values [mean±SEM]: 2.7±0.2μM vs. 11.3±1.2μM against 4T1 cells; 1.8±0.2 vs. 17.1±1.26μM against Miapaca-2 cells; p<0.0001). In both cell lines, CPA-LLP resulted in significantly lower clonogenicity than free CPA (p<0.05). Moreover, in both cell lines, CPA-LLP significantly enhanced the cell response to CCPM-(177)Lu radiotherapy as measured by clonogenic assay (p<0.05). In 4T1 and Miapaca-2 mouse xenograft models, the combination of CPA-LLP and CCPM-(177)Lu delayed tumor growth more than either monotherapy did alone. In the 4T1 tumor model, tumor size at 16days after treatment was significantly smaller with the combination therapy than with all the other treatments. In the Miapaca-2 model, the combination therapy resulted in the highest rate of mouse survival and prevented tumor relapse. In conclusion, the combination of CPA-LLP and CCPM-(177)Lu was an effective strategy for treating breast and pancreatic cancer and deserves further investigation.