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BackgroundTertiary lymphoid structures (TLSs) are immune aggregates in peripheral tissues that may support adaptive immune responses. Their presence has been associated with clinical response to checkpoint blockade therapy (CBT), but it is unknown whether TLS have prognostic significance independent of CBT in melanoma. We hypothesized that TLS in melanoma metastases would be associated with increased intratumoral lymphocyte infiltration, but that the intra-TLS immunological milieu would be distinct from the intratumoral immunological milieu. We also hypothesized that the presence of TLS would be associated with improved survival, and that TLS maturation or intra-TLS lymphocyte activity would also correlate with survival.MethodsCutaneous melanoma metastases (CMM) from 64 patients were evaluated by multiplex immunofluorescence for the presence and maturation status of TLS. Intra-TLS lymphocyte density, proliferation and B-cell Ig somatic hypermutation (AID+) were analyzed, as were markers of T-cell exhaustion and Th1/Tc1 differentiation. Associations between TLS maturation and intra-TLS immunologic activity were assessed, as well as associations with intratumoral immune cell infiltration. Independent associations with overall survival (OS) were assessed using log-rank tests and Cox proportional hazards models.ResultsTLS were identified in 30 (47%) of 64 CMM (TLS+) and were associated with increased intratumoral lymphocyte infiltration. However, proliferation of intra-TLS lymphocytes did not correlate with intratumoral lymphocyte proliferation. Most were early TLS; however, subsets of primary or secondary follicle-like TLS were also present. TLS+ lesions were associated with lower risk of tumor recurrence after metastasectomy and with improved OS in multivariate analyses (HR 0.51, p=0.04). OS was longer for TLS with low fractions of CD21+ B-cells (HR 0.29, p=0.02) and shorter for those with low AID+ fraction of B-cells (HR 2.74, p=0.03).ConclusionsThe presence of TLS in CMMs is associated with improved OS in patients treated with surgery before CBT, but TLS vary widely in maturation state, in proportions of proliferating T and B cells, and in markers of B cell function, including AID and CD21. Importantly, these features have additional prognostic significance, which suggest that some TLS may have regulatory function, while others functioning to support antigen-driven immune responses, depending on the cellular composition and activation status.

Background Broad implementation of model-based dose-finding methods, such as the continual reassessment method (CRM), has been limited, with traditional or modified 3 + 3 designs remaining in frequent use. Part of the reason is the lack of reliable, easy-to-use, and robust software tools for designing and implementing more efficient designs. Results With the aim of augmenting broader implementation of model-guided methods, we have developed a web application for the Bayesian CRM in the R programming language using the Shiny package. The application has two components, simulation and implementation. Within the application, one has the ability to generate simulated operating characteristics for the study design phase, and to sequentially provide the next dose recommendation for each new accrual or cohort based on the current data for the study implementation phase. At the conclusion of the study, it can be used to estimate the maximum tolerated dose (MTD). The web tool requires no programming knowledge, and it is free to access on any device with an internet browser. Conclusions The application provides the type of simulation information that aid clinicians and reviewers in understanding operating characteristics for the accuracy and safety of the CRM, which we hope will augment phase I trial design. We believe that the development of this software will facilitate more efficient collaborations within study teams conducting single-agent dose-finding trials.

Background With numerous and fast approvals of different agents including immune checkpoint inhibitors, monoclonal antibodies, or chimeric antigen receptor (CAR) T-cell therapy, immunotherapy is now an established form of cancer treatment. These agents have demonstrated impressive clinical activity across many tumor types, but also revealed different toxicity profiles and mechanisms of action. The classic assumptions imposed by cytotoxic agents may no longer be applicable, requiring new strategies for dose selection and trial design. Description This main goal of this article is to summarize and highlight main challenges of early-phase study design of immunotherapies from a statistical perspective. We compared the underlying toxicity and efficacy assumptions of cytotoxic versus immune-oncology agents, proposed novel endpoints to be included in the dose-selection process, and reviewed design considerations to be considered for early-phase trials. When available, references to software and/or web-based applications were also provided to ease the implementation. Throughout the paper, concrete examples from completed (pembrolizumab, nivolumab) or ongoing trials were used to motivate the main ideas including recommendation of alternative designs. Conclusion Further advances in the effectiveness of cancer immunotherapies will require new approaches that include redefining the optimal dose to be carried forward in later phases, incorporating additional endpoints in the dose selection process (PK, PD, immune-based biomarkers), developing personalized biomarker profiles, or testing drug combination therapies to improve efficacy and reduce toxicity.

Background: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. Methods: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. Results: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). Conclusions: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.

Background Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8 + T cell responses, whereas addition of an incomplete Freund’s adjuvant (IFA) would reduce magnitude and persistence of immune responses. Patients and methods Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). Results Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. Conclusions LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. Trial registration The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Introduction Infiltration of non-small-cell lung cancer (NSCLC) by CD8 + T lymphocytes predicts improved patient survival; however, heterogeneity of intratumoral localization complicates this assessment. Strategies for tumor sampling may not accurately represent the whole tumor. We hypothesized that sampling strategies may alter the identification of tumors with high CD8 density and affect the prognostic significance. Patients and methods Twenty-three primary NSCLC tumors were immunohistochemically stained for CD8 and were assessed using automated software with eight different sampling strategies or the whole tumor. Results of all sampling strategies were compared to the whole tumor counts (paired t tests, Pearson’s r). Associations between CD8 densities and overall survival were assessed (log-rank test). Results Counts from all eight sampling strategies significantly correlated with whole tumor counts ( p  ≤ 0.001). However, the magnitude of CD8 + cell counts and categorization into high vs low infiltrate groups were affected by the sampling strategy. The most concordant values were derived from random sampling of 20 % of the tumor, a simulated core biopsy, or from sampling the tumor center. TIL infiltration was associated with survival when sampling the center ( p  = 0.038), but not the invasive margin ( p  > 0.2) or other strategies. Conclusion Different tumor sampling strategies may yield discordant TIL density results and different stratification for risk assessment. Small biopsies may be particularly unrepresentative. Random sampling of larger tumor areas is recommended. Enumerating CD8 + T cells in the tumor center may have prognostic value.

BackgroundWe performed a clinical trial to evaluate safety and immunogenicity of a novel long peptide vaccine administered in combinations of incomplete Freund’s adjuvant (IFA) and agonists for TLR3 (polyICLC) and TLR7/8 (resiquimod). We hypothesized that T cell responses to minimal epitope peptides (MEPs) within the long peptides would be enhanced compared with prior vaccines with MEP themselves and that T cell responses would be enhanced with TLR agonists, compared with IFA alone.MethodsParticipants with resected stage IIB-IV melanoma were vaccinated with seven long melanoma peptides (LPV7) from tyrosinase, gp100, MAGE-A1, MAGE-A10, and NY-ESO-1, each containing a known MEP for CD8+ T cells, plus a tetanus helper peptide (Tet) restricted by Class II MHC. Enrollment was guided by an adaptive design to one of seven adjuvant combinations. Vaccines were administered at weeks 1, 2, 3, 6, 9, 12 at rotating injection sites. T cell and IgG antibody (Ab) responses were measured with IFN-gamma ELIspot assay ex vivo and ELISA, respectively.ResultsFifty eligible participants were assigned to seven study groups, with highest enrollment on arm E (LPV7+Tet+IFA+polyICLC). There was one dose-limiting toxicity (DLT) in Group E (grade 3 injection site reaction, 6% DLT rate). All other treatment-related adverse events were grades 1–2. The CD8+ T cell immune response rate (IRR) to MEPs was 18%, less than in prior studies using MEP vaccines in IFA. The CD8+ T cell IRR trended higher for IFA-containing adjuvants (24%) than adjuvants containing only TLR agonists (6%). Overall T cell IRR to full-length LPV7 was 30%; CD4+ T cell IRR to Tet was 40%, and serum Ab IRR to LPV7 was 84%. These IRRs also trended higher for IFA-containing adjuvants (36% vs 18%, 48% vs 24%, and 97% vs 60%, respectively).ConclusionsThe LPV7 vaccine is safe with each of seven adjuvant strategies and induced T cell responses to CD8 MEPs ex vivo in a subset of patients but did not enhance IRRs compared with prior vaccines using short peptides. Immunogenicity was supported more by IFA than by TLR agonists alone and may be enhanced by polyICLC plus IFA.Trial registration numberNCT02126579.

More complex research questions are being posed in early-phase oncology clinical trials, necessitating design strategies tailored to contemporary study objectives. This paper describes the proposed design of a Phase I trial concurrently evaluating the safety of a hematopoietic progenitor kinase-1 inhibitor (Agent A) as a single agent and in combination with an anti-PD-1 agent in patients with advanced malignancies. The study's primary objective was to concurrently determine the maximum tolerated dose (MTD) of Agent A with and without anti-PD-1 therapy among seven possible study dose levels. Our solution to this challenge was to apply a continual reassessment method shift model to meet the research objectives of the study. The application of this method is described herein, and a simulation study of the design's operating characteristics is conducted. This work was developed through collaboration and mentoring between the authors at the American Association for Cancer Research (AACR) and the American Society of Clinical Oncology (ASCO) annual AACR/ASCO Methods in Clinical Cancer Research Workshop. The aim of this manuscript is to highlight examples of novel design applications as a means of augmenting the implementation of innovative designs in the future and to demonstrate the flexibility of adaptive designs in satisfying modern design conditions. Although the design is presented using an investigation of Agent A with and without anti-PD-1 therapy as an illustrative example, the approach described is not specific to these agents and could be applied to other concurrent monotherapy and combination therapy studies with well-defined binary safety endpoints.

Background Most of the current designs used for Phase I dose finding trials in oncology will either involve only a single cytotoxic agent or will impose some implicit ordering among the doses. The goal of the studies is to estimate the maximum tolerated dose (MTD), the highest dose that can be administered with an acceptable level of toxicity. A key working assumption of these methods is the monotonicity of the dose–toxicity curve. Purpose Here we consider situations in which the monotonicity assumption may fail. These studies are becoming increasingly common in practice, most notably, in phase I trials that involve combinations of agents. Our focus is on studies where there exist pairs of treatment combinations for which the ordering of the probabilities of a dose-limiting toxicity cannot be known a priori. Methods We describe a new dose-finding design which can be used for multiple-drug trials and can be applied to this kind of problem. Our methods proceed by laying out all possible orderings of toxicity probabilities that are consistent with the known orderings among treatment combinations and allowing the continual reassessment method (CRM) to provide efficient estimates of the MTD within these orders. The design can be seen to simplify to the CRM when the full ordering is known. Results We study the properties of the design via simulations that provide comparisons to the Bayesian approach to partial orders (POCRM) of Wages, Conaway, and O'Quigley. The POCRM was shown to perform well when compared to other suggested methods for partial orders. Therefore, we comapre our approach to it in order to assess the performance of the new design. Limitations A limitation concerns the number of possible orders. There are dose-finding studies with combinations of agents that can lead to a large number of possible orders. In this case, it may not be feasible to work with all possible orders. Conclusions The proposed design demonstrates the ability to effectively estimate MTD combinations in partially ordered dosefinding studies. Because it relaxes the monotonicity assumption, it can be considered a multivariate generalization of the CRM. Hence, it can serve as a link between single and multiple-agent dosefinding trials.

Age has been evaluated as a prognostic factor in cervical cancer in both hospital- and population-based studies. Results regarding the relation of age and cervical cancer prognosis are conflicting. This study pursued a contemporary assessment of the association of extreme young age at the time of a cervical cancer diagnosis on survival. Institutional review board approval was obtained, and retrospective data collection at 2 academic institutions was performed. Inclusion criteria involved women ≤35 years diagnosed with cervical cancer between 1990 and 2012. Data included demographic and prognostic information pertinent to survival and progression. Characteristics of very young (≤25 years) and young (>25–35 years) women were compared. Kaplan-Meier estimates, the log-rank test, and Cox proportional hazards modeling were used to assess the association of age, tumor histology, grade, stage, and parametrial involvement with progression-free survival (PFS) and overall survival (OS). Incident cases (n = 126) of cervical cancer in patients ≤35 years of age were identified of which complete clinical information was available for 114 women. Fifteen percent (17 of 114) were ≤25 years, with the remaining 85% (97 of 114) being 26 to 35 years of age. Race, smoking status, and marital status were comparable between the 2 groups. Squamous histology dominated overall (77 of 114; 68%) with adenocarcinoma contributing ~25% (30 of 114; 26%) of cases. The majority (96 of 114, 84%) had either stage 1A (31 of 114, 27%) or 1B (65 of 114, 57%) disease. A log-rank test revealed no evidence to infer a difference in either PFS or OS among the age groups (P = 0.511 and P = 0.340). In a univariate analysis, grade and stage significantly affected OS (P < 0.0001, P = 0.045), and stage significantly affected PFS (P < 0.0001). In multivariate modeling, presence of parametrial involvement and histologic cancer type significantly affected both PFS (P = 0.002, P = 0.001) and OS (P = 0.001, P = 0.001). Tumor histology, parametrial involvement, and stage continue to be strong prognosticators for PFS and OS. Progression and survival outcomes are age independent in women with cervical cancer ≤35 years of age. Further study of a larger young cohort may potentially yield different outcomes.