Any data analysis carried out independently by the investigator must be approved by the statistician before publication or presentation. 15.1 Study Design Overview This single -arm phase II study will assess the impact of tetanus pre- conditioning and adjuvant GM-CSF on overall survival of newly diagnosed GBM patients who have undergone definitive resection , are CMV+ and unmethylated, and completed standard temoz olomide and radiation treatment . After completing standard of care radiotherapy with concurrent temozolomide, patients will receive 1 cycle of dose intensified TMZ followed by pp65 DC vaccination beginning on day 23. 15.2 Analysis Sets All patients who initiate dose -intensified TMZ after completion of standard of care RT and TMZ will be inclu ded in the primary efficacy analyses, and toxicity summaries. I-ATTAC Version: 20220204 47 Subject to the availability of data, analyses of correlative objectives will include all or some of the patients who undergo vaccine 4. For analyses of migration, the first 16 patients who rea ch vaccine 4 will undergo migration analyses . For CCL3, all patients who reach vaccine 4 will be included in statistical analyses. For analyses of polyfunctionality, the first 24 patients who reach vaccine 4 will be considered. 15.3 Patient Demographics and Other Baseline Characteristics Summaries of clinical and socio -demographic characteristics will be generated within four groups of patients: (1) All patients who sign informed consent prior to the initiation of standard of care temozolomide and radiation, (2) All patients who start dose intensified TMZ, (3) All patients who receive at least one pp65 DC vaccination , and (4) All patients who receive vaccine 4. Categorical descriptors will be summarized using frequency distributions; whereas, interval varia bles will be summarized using percentiles, as well as means and standard deviations. 15.4 Treatments Among patients who initiate dose intensified TMZ, a frequency distribution will be generated for the number of vaccines received by each patient. 15.5 Primary Objective The primary objective of this study is to determine if Td preconditioning of pp65 loaded DC vaccination with GM -CSF extends survival relative to a historical benchmark. 15.5.1 Variable Overall Survival (OS) is defined as the time between initiation of dose -intensified TMZ and death, or last follow -up if the patient remains alive at the time of analysis. 15.5.2 Statistical Hypothesis, Model, and Method of Analysis The Kaplan -Meier estimator will graphically describe OS. Median OS will be estimated , with 95% confidence intervals , as well as 12, 18, 24, 36, and 48- month survival rates. A one -sample logrank test [124, 125] will compare this curve to that reported by Gilbert [ 126] in which the median OS from the start of adjuvant temozolomide treatment was 14.0 months (95% CI: 12.9 t o 14.7 months) for patients with newly diagnosed GBM that are unmethylated. A n intent -to-treat approach will be used in analyses . The statistical hypothesis and power calculations are provided in Section 15.9. Given the possibility that the characteristics of patients who have enrolled on this protocol differs from that described by Gilbert [127] for RTOG 0825, additional analyses will be conducted using the nomogram published by Gittlemen [ 128] for RTOG 0525 and 0825. For each patient, the probability of 24- month survival will be estimated from the nomogram based upon the patients ’ baseline characteristics. From these individualized estimates of 24 -month survival, an overall estimate of 24 -month survival will be generated with 95% confidence i nterval. That confidence interval will be compare to the actual 24 -month survival confidence interval observed. 15.5.3 Handling of Missing Values, Censoring , and Discontinuation I-ATTAC Version: 20220204 48 Patients who withdraw from the study without an allowance for survival follow -up, or are lost to follow -up will have OS censored at the date that the patient was last know alive. 15.6 Secondary Objectives This study has five secondary objectives. The first 3 secondary objectives examine the impact of functional measures of CMV pp65- loaded D C vaccine efficacy on survival post -vaccine. These measures include migration (Section 15.6.1) , fold change in CCL3 (Section 15.6.2) , and fold change in pp65 antigen- specific polyfunctionality (Section 15.6.3) . Power calculations will be provided for each of the secondary objectives. Power calculations for the three functional predictors of survival post -vaccine 4 will be adjusted to account for multiple comparisons using a Bonferroni correction, and will used a type I error rate of 0.0167. The other secondary objectives include an assessm ent of whether the percent of T Reg of CD4+ T cells remains stable or decreases without additional temozolomide after vaccine 1 (Section 15.6.4), and an assessment of the safety of the protocol’s treatment regimen (Section 15.6.5). 15.6.1 Secondary Objective #1: Migration and Survival PLEASE NOTE: The study originally planned to address secondary objective #1 as descr ibed below. However, with the activation of Protocol v.20220204, the plans for examining the relationship between migration and survival have been dropped. Objective To assess the association between migration of CMV pp65 RNA -pulsed DCs with GM -CSF to si te-draining inguinal lymph nodes after Td pre -conditioning and survival after vaccine # 4 . Analysis Set All patients who undergo migration studies at vaccine 4 . Due to budgetary considerations, only the first 16 patients who reach vaccine 4 will undergo migration studies . Outcome Survival from vaccine 4 . Hypothesis Patients with greater migration will have longer survival . Analysis Plan The Cox proportional hazards model will assess the impact of migration on survival after vaccine #4 . Migration is de fined as the maximum percentage of 111In-labeled DCs reaching inguinal nodes during the 48 hours after the 4th vaccination . The hazard ratio associated with a 1 -unit change in migration will be estimate d with 95% confidence intervals. Power Calculations Cox proportional hazards model will assess the impact of peak migration at 24 or 48 hours after vaccine 4 on subsequent survival, defined as the time between vaccine 4 and death, or last follow -up if the patient remains alive at the time of analysis. Data from ATTAC [ 129] show that the standard deviation (SD) for peak migration is 7.86 and t hat the hazard ratio associated with 1 unit increase in migration is approximately 0.86 . Assuming SD=7.86, there would be 80% power to detect a hazard ratio of 0.86 assuming a two -tailed test (α=0.0167) within a Cox model after 8 deaths [130, 131]. Assuming the median OS post -vaccine 4 is 24 months, approximately 24 months after the 16th patient undergoes migration studies will 8 deaths be observed. I-ATTAC Version: 20220204 49 15.6.2 Secondary Objective #2: CCL3 and Survival Objective To assess the association between CCL3 measured post-Td administration and survival after vaccine # 4 . Analysis Set All patients who undergo assessment of CCL3 after Td administration. All patients who reach vaccine 4 when Td is administered will be included in analyses. Forty (40) patients are anticipated . Outcome Survival from vaccine 4 . Hypothesis Greater levels of CCL3 are associated with longer survival . Analysis Plan The Cox proportional hazards model will assess the impact of CCL3 on survival post-vaccine 4 . The hazard ratio associate with a 1 -unit increase in CCL3 will be estimated with 95% confidence intervals. Power Analyses Cox proportional hazards model will assess the impact of CCL3 on survival post - vaccine 4 . Within ATTAC [129], the standard deviation for CCL3 was 12.9 and the hazard ratio for survival post -vaccine 4 was approximately 0.95 for a 1 unit increase in CCL3 . Assu ming SD=12.9, there would be 80% power to detect a hazard ratio of 0.95 with a two- tailed test (α=0.0167) within a Cox model under these assumptions after 24 deaths [130, 131]. If we assume that the median OS post-vaccine 4 is 24 months, then 24 deaths should be observed approximately 20 months after the 40th patient receives vaccine 4. 15.6.3 Secondary Objective #3: Polyfunctionality and Survival Objective To assess the assoc iation between fold changes in T cell polyfunctionality from baseline to pheresis 2 , and survival after vaccine #4 . Analysis Set All patients who have a baseline (pre vaccine 1) and polyfunctionality assessed at leukapheresis 2 who receive vaccine # 4. P olyfunctionality is assessed at leukapheresis so that results do not reflect the impact of Td- preconditioning post- vaccine 4 assessment of polyfunctionality . Due to budgetary considerations, only the first 24 patients who reach vaccine 4 will be analyzed. Outcome Survival from vaccine 4 . Hypothesis Greater fold changes in polyfunctionality are associated with longer survival . Analysis Plan The mean fold change from baseline to leukapheresis 2 will be summarized for the frequency of pp65 antigen- specific CD8+ T cells producing three or more cytokines (IFNγ, CCL3, IL -2, TNFα, CD107a). Cox proportional hazards model will assess the association between fold change increase between baseline and the leukapheresis 2 in the frequency of pp65 antigen- specific CD8+ T cells producing three or more cytokines (IFNγ, CCL3, IL -2, TNFα, CD107a), and survival post-vaccine 4 . The hazard ratio associate with a 1 -unit fold change in polyfunctionality will be estimated with 95% confidence intervals. Power Analyses Cox propo rtional hazards model will assess the impact of fold change of pp65 - specific polyfunctional CD8+ T cells between baseline and leukapheresis 2 on survival after vaccine #4 . Data from Pro00000580 ERADICATE [132] shows that the SD for fold change in polyfunctionality among vaccinated patients is 1.589, and that the hazard ratio associated with 1 unit increase in fold change is I-ATTAC Version: 20220204 50 approximately 0.609. Assuming SD=1.589 , there would be 80% power to detect a hazard ratio of 0.6 assuming a two- tailed test (α=0.0167) within a Cox model after 16 deaths [130, 131]. If we assuming that the median OS post -vaccine 4 is 24 months, then 16 deaths should be observed approximately 38 months after the 24th patient receives vaccine 4. Additional Analyses Many of the 24 patients who have assessments at pre -vaccine 1 and leukapheresis 2 will also have an assessment of polyfunctionality at pre -vaccine 6. Among those patients who have all 3 assessments, a paired comparison of the fold change in polyfunctionality at pheresis 2 and vaccine 6 will be conducted using either a paired t -test or a Wilcoxon signed rank test. These analyses will assess whether levels of polyfunctionality are maintained in the absence of DI -TMZ. In addition, the Wilcoxon rank sum test will assess whether significant fold changes occur at pheresis 2 . 15.6.4 Secondary Objective # 4: T Regs Objective To assess whether percent T Reg levels increase further after vaccine 1 Analysis Set All treated patients Outcome Maximum peak increase from vaccine 1 in percent T Reg of CD4+ T cells Hypothesis With no additional temozolomide treatment afte r vaccine 1, the level of percent TReg will not significantly increase above vaccine 1 levels. Rather there should be no change or a reduction in T Reg levels. Analysis Plan A one -tailed t -test will be conducted to assess whether the mean for the maximu m change from vaccine 1 in percent T Reg is significantly greater than 0. If the mean change is less than 0.71 (i.e. near 0 or a decrease), we will infer that levels of percent T Reg do not increase after vaccine 1. Rather, they are either stable or decrea se. Power Analyses In ATTAC -GM, the mean change in the percent T Reg of CD4+ T cells between leukapheresis and vaccine 1, a period during which DI -TMZ is administered, wa s 5.77 (SD=4.52). Between vaccine 1 and the next cycle of DI -TMZ, the peak mean increase in percent T Reg from vaccine 1 was -1.104 (SD=1.65). Assuming 48 patients and SD=1.65 for the mean change from vaccine 1 within I-ATTAC , there will be 90% power to detect an increase from vaccine 1 in the percent of T Reg if the true mean increase is 0.71 or greater assuming a one -tailed test (α=0.05) . Additional Analyses If the primary analysis that examines the maximum increase from vaccine 1 percent T Reg of CD4+ T cells observed at any point after the initial vaccine detects such an increase, the timing of that increase is of interest. Hence, we will also examine the change from vaccine 1 to each of the follow - up assessments (leukapheresis 2, vaccine 4, vaccine 5, and vaccine 6). A generalized linear model may be used to explore these longitudinal changes. We may also use one -tailed t-tests to describe these changes. The change between vaccine 1 to leukapheresis 2 reflects the impact of vaccine 1, 2 and 3; whereas the change at vaccine 4 reflects the effect of the first 3 vaccines as well as T d pre-conditioning. I-ATTAC Version: 20220204 51 We will also conduct a t -test to examine the change in T Reg levels observed before DI-TMZ and vaccine 1 to confirm that T Reg levels are elevated with DI -TMZ, as well as the change between leukapheresis 1 and initiation of DI- TMZ. 15.6.5 Secondary Objective # 5: Toxicity Objective Assess the safety of DI -TMZ treatment followed by DC vaccination . Analysis Set All patients who initiate DI -TMZ treatment. Outcome Proportion of patients with unacceptable toxicity as defined in Section 9.1.1 . Hypothesis The toxicity profile associated with DI -TMZ followed by DC vaccination is safe. Monitoring Plan Table A: Toxicity Monitoring Rules Number of patients accrued Number of patients with unacceptable toxicity requirin g accrual suspension ≤5 ≥2 6-11 ≥3 12-17 ≥4 18-23 ≥5 24-29 ≥6 30-34 ≥7 35-39 ≥8 ≥40 ≥9 Given that both long - and short -term toxicities are of interest in this study, it is not feasible to suspend accrual while toxicity is assessed as is often done in phase I trials. If the criteria in Table A are satisfied or there are other reasons for concern about the safety of patient treatment (e.g. , treatment -related toxic death), accrual will be suspended and data will be carefully reviewed to determine if accrual should be per manently terminated or the protocol modified. Table A provides conditions under which accrual will be temporarily suspended and data carefully reviewed to determine the appropriate action, including permanent study termination, continuation with patient a ccrual after appropriate amendment, or continuation with patient accrual with no modification of the protocol. These guidelines have not been adjusted for differential length of follow-up of accrued patients. If a death occurs within 30 days of DC vacci ne administration, that is not attributable to progressive disease or other obvious non- study related cause (i.e. motor vehicle accident), enrollment of new subjects and all vaccinations will be suspended until review by the PI and IND sponsor has been com pleted and the FDA notified. If attribution of death is determined by the PI, IND sponsor or FDA to be possibly, probably, or definitely related to trial drug, then continued treatment of all enrolled subjects with trial drug will be suspended until the review is completed and recommendations for the study continuation have been I-ATTAC Version: 20220204 52 issued and approved by FDA. Any death attributed to trial drug, regardless of the timeline with respect to last treatment, will result in suspension of enrollment and suspension of continued treatment for enrolled subjects until review by FDA is complete and recommendations for study continuation issued and approved. TABLE B: Probability of Accrual Suspension Underlying unacceptable toxicity Rate Probability of accrual suspension 0.05 0.036 0.10 0.200 0.15 0.502 0.20 0.784 0.25 0.936 0.30 0.987 The probability of accrual suspension as a function of the true unacceptable toxicity rate is provided in Table B. These statistics were generated assuming toxicity outcome was known at the time of accrual, and ignored issues such as time to toxicity, accrual rate, and length of follow -up. Every 6 months from the time the first patient received vaccine #1, the toxicity experienced by patients accrued to this study will be summarized and reviewed regardless of the number of patients accrued to determine whether the overall toxicity profile of treatment is unacceptable or not. Analysis Plan The proportion of patients who experience an unacceptable toxicity as defined in Section 9.1.1 Additional Analyses Adverse events will be summarized in several other forms to satisfy scientific and monitoring needs, as well as various regulatory reporting needs (e.g. FDA, DCI S afety Oversight Committee, and ClinicalTria ls.gov). For the se summaries , the frequency of adverse events will be tabulated by the maximum grade experienced. 15.7 Exploratory Objectives This study has 3 exploratory objectives: (1) To assess the relationship between migration and serum CCL3, (2) To assess the relationship between pp65- specific polyfunctional CD8+ T cells and migration, and (3) To assess the relationship between pp65- specific polyfunctional CD8+ T cells and serum levels of CCL3. 15.7.1 Exploratory Objective #1: Migration and CCL3 Objective To assess the relationship between serum CCL3 levels and increased DC migration . Analysis Set All patients who undergo migration studies who have CCL3 levels measured . Outcome Correlation coefficient between fold change of CCL3 and migration . Hypothesis We hypothesize that fold change in CCL3 will be highly correlated with increased migration. I-ATTAC Version: 20220204 53 Analysis Plan A Spearman rank correlation coefficient will assess the association between CCL3 change from pre to post -vaccine 4, and migration. Additional Anal yses As an additional exploratory analysis of CCL3, a one -sample t -test or a Wilcoxon rank sum test will assess whether fold change in CCL3 between pre - and post - Td pre -conditioning differs from 0. 15.7.2 Exploratory Objective #2: Polyfunctionality and Migrati on Objective To assess the relationship between pp65 -specific polyfunctional CD8+ T cells and DC migration . Analysis Set All patients who undergo migration studies who have polyfunctional levels measured . Outcome Correlation coefficient between fold cha nge in polyfunctionality and migration Hypothesis We hypothesize that increased pp65 -specific polyfunctionality CD8+ T cells will be highly correlated with increased DC migration. Analysis Plan A Spearman rank correlation coefficient will assess the asso ciation between fold change in polyfunctionality and migration. 15.7.3 Exploratory Objective #3: Polyfunctionality and CCL3 Objective To assess the relationship between pp65 -specific polyfunctional CD8+ T cells and serum CCL3 . Analysis Set All patients who u ndergo polyfunctional studies and have CCL3 levels measured. Outcome Correlation coefficient between fold change in polyfunctionality and fold change in CCL3 . Hypothesis We hypothesize that increased pp65 -specific polyfunctionality CD8+ T cells will be highly correlated with increases in serum CCL3 after Td preconditioning. Analysis Plan A Spearman rank correlation coefficient will assess the association between fold change in polyfunctionality and fold change in CCL3. 15.8 Interim Analysis As described in S ection 15.9, we anticipate that at the time of the primary study analysis 33 of the patients treated with DI -TMZ will have died. An interim analysis for futility will be conducted after approximately 17 patients h ave died, assuming that occurs before accrual is completed. The α-spending function that approximately an O’Brien- Fleming boundary will defined critical values that will be used in making that assessment [ 133]. Based upon these analyses and available correlative data, a decision will be made concerning continued accrual without modification, continued accrual with modifications to the protocol, or termination of patient accrual. 15.9 Sample Size Calculation Approximately 64 patients will be accrued to this study, with the goal of initiating DI -TMZ in 48 of these patients . To evaluate the observed OS within this study of pp65 DC vaccination with tetanus pre - conditioning and adjuvant GM -CSF, we will consider Gilbert’s study of dose -dense temozolomide for newly diagnosed glioblastoma. Gilbert reports a median OS from initiation of adjuvant I-ATTAC Version: 20220204 54 treatment among methylated patients to be 14.0 months (95% CI: 12.9 to 14.7 mont hs). Though the median OS from initiation of adjuvant DI -TMZ treatment in the ATTAC -GM pilot is 37.7 months (95% CI: 18.2, ∞) , we hypothesize for power calculations that the true median OS for our novel treatment regimen is approximately 24 months. Ass uming accrual of 64 patients over a period of 24 months, 28 months follow -up after the last patient initiates adjuvant treatment, and 12 patients who initiate vaccine treatment, a two -sided, a one -sample logrank test has 80% power at a 0.05 significance level to detect a hazard ratio of 0.6125 (i.e. = 14.7 / 24) when the median survival of the historic control group is 14.7 months, the upper bound of the 95% confidence interval for median survival reported by Gilbert [124, 125, 131] . We anticipate that at the time of the primary study analysis 3 3 of these pati ents will have died. 16 ADMINISTRATIVE AND E THICAL CONSIDERATIONS 16.1 Regulatory and Ethical Compliance This protocol was designed and will be conducted and reported in accordance with the International Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice, the Declaration of Helsinki, and applicable federal, state, and local regulations. 16.2 DUHS Institutional Review Board and DCI Cancer Protocol Committee The protocol, informed consent form, advertising material, and addit ional protocol -related documents must be submitted to the DUHS IRB and DCI Cancer Protocol Committee ( CPC) for review. The study may be initiated only after the Principal Investigator has received written and dated approval from the CPC and IRB. The Princ ipal Investigator must submit and obtain approval from the IRB for all subsequent protocol amendments and changes to the informed consent form. The CPC should be informed about any protocol amendments that potentially affect research design or data analysi s (i.e. amendments affecting subject population, inclusion/exclusion criteria, agent administration, statistical analysis, etc.). The Principal Investigator must obtain protocol re -approval from the IRB within 1 year of the most recent IRB approval. The P rincipal Investigator must also obtain protocol re -approval from the CPC within 1 year of the most recent IRB approval, for as long as the protocol remains open to subject enrollment. 16.3 Informed Consent The informed consent form must be written in a manner t hat is understandable to the subject population. Prior to its use, the informed consent form must be approved by the IRB. The Principal Investigator or authorized key personnel will discuss with the potential subject the purpose of the research, methods, potential risks and benefits, subject concerns, and other study - related matters. This discussion will occur in a location that ensures subject privacy and in a manner that minimizes the possibility of coercion. Appropriate accommodations will be made avail able for potential subjects who cannot read or understand English or are visually impaired. Potential subjects will have the opportunity to contact the Principal investigator or authorized key I-ATTAC Version: 20220204 55 personnel with questions, and will be given as much time as nee ded to make an informed decision about participation in the study. Before conducting any study -specific procedures, the Principal Investigator must obtain written informed consent from the subject. The original informed consent form will be stored with the subject’s study records, and a copy of the informed consent form will be provided to the subject. The Principal Investigator is responsible for asking the subject whether the subject wishes to notify his/her primary care physician about participation in the study. If the subject agrees to such notification, the Principal Investigator will inform the subject’s primary care physician about the subject’s participation in the clinical study. 16.4 Privacy, Confidentiality, and Data Storage The Principal Investigato r will ensure that subject privacy and confidentiality of the subject’s data will be maintained. Research Data Security Plan s (RDSPs ) will be approved by the appropriate institutional Site Based Research group. To protect privacy, every reasonable effort will be made to prevent undue access to subjects during the course of the study. Prospective participants will be consented in an exam room where it is just the research staff, the patient and his family, if desired. For all future visits, interactions wit h research staff (study doctor and study coordinators) regarding research activities will take place in a private exam room. All research related interactions with the participant will be conducted by qualified research staff who are directly involved in t he conduct of the research study. To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock and key accessible only by the research staff. Subjects will be identified only by a unique study number and subject i nitials. Electronic records of subject data will be maintained using a Clinical database, which is housed by the DCI. Access to electronic databases will be limited to the Principal Investigator, key personnel, statisticians, the Radiolabeled Pharmacy pers onnel, and the PRTBTC data manager. Data stored on portable memory devices will be de -identified. The security and viability of the IT infrastructure will be managed by the DCI and/or Duke Medicine. Upon completion of the study, research records will be archived and handled per DUHS Human Research Protection Program (HRPP ) policy. Subject names or identifiers will not be used in reports, presentations at scientific meetings, or publications in scientific journals. 16.5 Data and Safety Monitoring Data and Saf ety Monitoring will be performed in accordance with the DCI Data and Safety Monitoring Plan. For a more detailed description of the D SMP for this protocol, refer the separate upload in e lectronic IRB. 16.6 Protocol Amendments All protocol amendments must be ini tiated by the Principal Investigator and approved by the IRB prior to implementation. IRB approval is not required for protocol changes that occur to protect the safety of a subject from an immediate hazard. However, the Principal Investigator must inform the IRB and all other applicable regulatory agencies of such action immediately. I-ATTAC Version: 20220204 56 Though not yet required, the CPC should be informed about any protocol amendments that potentially affect research design or data analysis (i.e., amendments affecting subject population, inclusion/exclusion criteria, agent administration, etc.). 16.7 Records Retention The Principal Investigator will maintain study -related records for the longer of a period of: - at least two years after the date on which a New Drug Application is app roved by the FDA - at least two years after formal withdrawal of the IND associated with this protocol - at least six years after study completion (Duke policy). 16.8 Conflict of Interest The Principal Investigator and Sub- Investigators must comply with applicable federal, state, and local regulations regarding reporting and disclosure of conflict of interest. Conflicts of interest may arise from situations in which financial or other personal considerations have the potential to compromise or bias professional judg ment and objectivity. Conflicts of interest include but are not limited to royalty or consulting fees, speaking honoraria, advisory board appointments, publicly - traded or privately -held equities, stock options, intellectual property, and gifts. The Duke U niversity School of Medicine’s RIO reviews and manages research -related conflicts of interest. The Principal Investigator and Sub- Investigators must report conflicts of interest annually and within 10 days of a change in status, and when applicable, must have a documented management plan that is developed in conjunction with the Duke RIO and approved by the IRB/IEC. 16.9 Registration Procedure After patients have been enrolled, subject registration will be entered into the Duke eResearch system and the subject’s visits associated in the Duke Epic Maestro Care system with this protocol which is entered after Duke IRB approval . I-ATTAC Version: 20220204 57 17 REFERENCES 1. Kelly, P.J., Stereotactic resection and its limitations in glial neoplasms. Stereotact Funct Neurosur g, 1992. 59(1 -4): p. 84 -91.