Patient demographics and the results of CRS/HIPEC are outlined in Table 1. The 46 patients included 15 females and 31 males with a mean age of 55 years. Twenty-four patients had appendiceal peritoneal metastases, 20 patients had colorectal peritoneal metastases, and 2 patients had peritoneal mesothelioma. Overall, 34 (74%) patients had recurrent disease at presentation, 32 (70%) had undergone previous CRS, and 20 (43%) had previously undergone HIPEC. On average, patients had been diagnosed with peritoneal metastases for 27 months prior to being recruited into the trial. The median Peritoneal Cancer Index (PCI) was 15, indicating a high burden of intraperitoneal disease. CC-0 was achieved in 35 patients, 9 patients had small-volume residual disease (CC-1), and 2 patients had moderate residual disease (CC-2).
Vaccine production and administration data are outlined in Table 2. All patients had tumor harvested for antigen loading of DCs. The goal was for recovery of at least 8 g of tumor and for recovery of 1.2 × 107 viable tumor cells for optimal loading of DCs. Based on viable cell count, excluding white blood cells, we planned to load the DCs at a target ratio of 30:1 monocytes to tumor cells (corresponding to a target ratio of 101:DC to tumor cells). Overall, we were able to harvest a median of 37.5 g of tumor, ranging from 2 to 71 g. While we did not use CD326 (EpCAM) staining to determine the numbers of tumor cells (vs. stromal cells) for DC antigen loading, we did measure it in retrospect for patients with colon or appendiceal tumors to differentiate the tumor cells from stromal cells and estimate the number of tumor cells used for DC loading. A median of 1.8 × 106 viable, EpCAM + tumor cells were recovered for DC loading (ranging from 3.9 × 106 to 3.5 × 107). Only eight patients had the desired number (1.2 × 107) of EpCAM + tumor cells for DC loading. While we intended to load DCs with tumor-derived cells at a ratio of 30:1, when analyzing EpCAM + staining in retrospect, we actually loaded with a median ratio of 15 times lower (when calculating ratios of DCs to EpCAM + tumor cells, range 0.12–28). No patients reached the desired 30:1 ratio of DC precursor monocytes to EpCAM + tumor cells (or the target 10:1 ratio of DCs to tumor cells).
DCs were able to be isolated and matured, with 97% of αDC1 expressing HLA-DR and CD86. CD40L-induced IL-12p70 production was, on average, 2.8 ± 3.9 ng/mL/2 × 104 αDC1/24 h, indicating adequate maturation.9 Fifty-four percent of patients had > 1.0 ng/mL/24 h IL-12p70. Production > 3.0 ng/mL has been previously correlated with clinical response, and 10 patients had IL-12p70 levels > 3.0, ranging up to 17.20
Because of low tumor cell recovery from operative tissue specimens, we did not always have enough primed αDC1 for optimal vaccination dosing and/or for completing the priming and three booster doses. This resulted in only 9 of the 46 patients receiving the target dose of 3 × 106 αDC1, and only 32 of the 46 patients received all three boosters—6 received two boosters, 7 received only one booster, and 1 patient received only the initial priming dose of DCs. Only 8 of the 46 patients received all three boosters at the target dose.
Summary of Adverse Events
Grade 1 flu-like symptoms were the most common AEs attributed to the DC vaccination and CKM, including chills (n = 36), fatigue (n = 19), and nausea (n = 19). They were usually self-limited to 24 h after each vaccine dose. Grades 1 and 2 injection-site reactions were relatively uncommon, affecting only two patients. Six patients had grades 3–5 AEs, possibly attributed to treatment. These included a decrease in lymphocyte count, diarrhea, transient blurry vision, stroke, heart failure, and gastrointestinal bleed while receiving blood thinners for personal history of stroke (Table 3). One patient experienced transient blurry vision that may be related to rintatolimod and was self-limited.
Serum Cytokines and Chemokines
We measured CXCL10, CXCL11, CXCL12, IL-6, IL-8, IL-10, and TNFα in serum at baseline and after the first booster with vaccine and CKM. All these inflammatory cytokines demonstrated an increase in serum concentrations after CKM treatment (Table 4).
The study was originally powered to determine a 25% improvement in PFS over historical controls from our own database, stratified by histology: colon cancer, appendiceal cancer, and mesothelioma with an accrual goal of 168 patients. The expected and target median PFSs were 20 and 25 months for appendiceal cancer, 12 and 15 months for colorectal cancer, and 20 and 25 months for peritoneal mesothelioma, respectively. The trial was terminated early after an interim analysis demonstrated futility due to a lack of improvement in PFS (colon cancer), variable tumor grade affecting results (appendiceal), or slow accrual (mesothelioma). In total, 46 patients were enrolled and had evaluable data prior to early termination.
PFS for colon cancer was 20.5 months for moderately differentiated tumors, and 8.9 months for poorly differentiated tumors (Fig. 2a). Two of 11 patients with moderately differentiated tumors have not recurred at 58 and 46 months after CRS/HIPEC. Both had PCI scores of < 12 and had all gross disease removed at the time of surgery. Overall survival for the moderately differentiated tumors was not reached, and for poorly differentiated tumors was 18.3 months (Fig. 2b). This is consistent with our prior reports of CRS/HIPEC for colon peritoneal metastases.
For appendiceal cancers, PFS was 50.4 months for low-grade (grade G1), 34.2 months for intermediate-grade (grade G2), and 8.9 months for high-grade/poorly differentiated/signet ring cell cancers (grade G3) (Fig. 3a). Three of eight patients with grade G2 tumors have not recurred at 55, 44, and 35 months of follow-up, with PCI scores of 8, 3, and 28, respectively, and all gross disease removed. Four of seven patients with grade G1 tumors have not recurred at 55, 51, 49, and 45 months, with PCI scores of 10, 14, 16, and 10, respectively, and all gross disease removed. Three of eight patients with grade G3 tumors have died of their disease at 6, 7, and 8 months after surgery; the remaining five patients are still alive 30–46 months after surgery. One of eight patients with grade G2 tumors had died at 44 months, with the remaining seven patients were alive 35–56 months after surgery. All patients with grade G1 tumors were still alive at a median follow-up of 50 months (Fig. 3b). These results are consistent with our previously reported results of survival for appendiceal cancer after CRS/HIPEC. The two patients with mesothelioma recurred at 25 and 8 months and died of their disease at 40 and 10 months, respectively. Both had biphasic mesothelioma, with PCI scores > 20 and complete cytoreduction.
As discussed above, 10 patients had IL-12p70 production > 3 ng/mL/24 h. No trend towards improved survival could be identified for those patients (data not shown). Two patients received all intended target-dose priming and booster injections of αDC1, with a high percentage of EpCAM+ cell loading. The first patient had high-grade appendiceal cancer, with a PCI score of 21 and minimal residual gross disease after CRS. He received all four doses of vaccine and three doses of CKM, and had 25.7% EpCAM+ cells loaded at a ratio of 30:1, with IL-12p70 production of 2.3 ng/mL/24 h. Unfortunately, the patient developed progressive disease 4.9 months after surgery and died of his disease 7.4 months after surgery. The second patient had poorly differentiated colon cancer, with a PCI score of 20 and no residual disease after CRS. He had 34.1% EpCAM+ cells and IL-12p70 production of 4 ng/mL/24 h. He developed a recurrence at 3.2 months and died 18 months after surgery.