Colorectal cancer is a leading cause of cancer-related deaths worldwide. Novel drugs have been approved for the treatment of chemo-refractory metastatic colorectal cancer (mCRC) patients, however, despite their clinical approval, the success rates are highly variable. RENCA Macrobeads (RMBs), a form of biological-systems based therapy for the treatment of mCRC has been tested in a clinical trial setting. RMBs adapt to the cancerous environments through the regulation of biochemical pathways and the release of anti-neoplastic factors leading to a positive therapeutic response and improved survival in mCRC patients. We report here phase II mCRC studies final results. We have compared prospectively the safety and efficacy between responder (R) and non-responder (NR) patients with mCRC refractory to standard therapies who were implanted with RENCA macrobeads (8 RMBs/kg body weight) in a laparoscopic setting (n=75). Overall survival (OS) using confidence intervals (95% CIs); Tumor Markers Response defined as a ≥ 20% decrease from baseline in one or both of CEA or CA 19-9; Disease Control Rate (DCR), and Objective Response Rate (ORR) were assessed to confirm effectiveness of the RMB implantation approach to the treatment of mCRC. Seventy-five metastatic CRC patients (34 phase IIa; 41 phase IIb) were treated with RENCA macrobeads, in a laparoscopic setting, after the failure of standard therapies. Post-implantation 1, we observed tumor marker response in 48/75 patients (64%) on the time period up to and including day 30; Undetectable response was marked in 27/75 patients (36%) [baseline median values R=60.1 ng/mL vs. NR=63.3 ng/mL; D14 R=69.3 ng/mL vs. NR=68.6 ng/mL; D30 R=47.5 ng/mL vs. NR=72.7 ng/mL]. Median OS defined as the interval from the screening date pre-RMB treatment to the first documented progression or death from any cause, was 7.7 months (95% confidence interval [CI], 6.1 - 12.2 months) for IIa mCRC patients, and 9.25 months (95% confidence interval [CI], 6.03 - 11.44 months) for the phase IIb group, excluding 5 patients who were alive and doing well at the time of data lock. OS between responders and non-responders was significant [median R=9.1 vs. NR=3.9 months; p = 0.004]. The DCR (stable response rate) was 20%, and the ORR was 59%. The RENCA Macrobeads were well tolerated. The most common adverse reactions were abdominal pain, fatigue, and pyrexia - majority related to transient SIRS response post laparoscopic procedure. In phase IIa and IIb studies we have shown that the RMB implantation is feasible, safe and may offer clinical benefit in metastatic CRC patients. Further analyses to confirm the above data will be conducted in a randomized phase III trial setting.

Introduction: Additional options are needed for metastatic colorectal patients who despite all current standard treatments progress. The RENCA Macrobead therapy represents an entirely new concept in cancer - a biological-systems approach that uses mouse renal adenocarcinoma cells entrapped in double agarose macrobeads. These inhibit the growth of mCRC by different pathways because of the array of signals they release (MEF-2, 40% of the effect). Reported here are further survival and PET-CT findings/evaluation of tumor response from the open-label, multi-site Phase IIb study. Methods: The population in this study included forty-one patients with documented mCRC refractory to standard therapies. They received eight RMBs/kg of body weight through a laparoscopic procedure up to four times in this Phase IIb trial. Post-implant patients were monitored at Days 14, 30, 60 and 90. Efficacy and safety were assessed with imaging, lab profile and physical exams. Tumor markers: CA 19-9 and/or CEA were tested for correlation between Responders (≥20% decrease) and Non-Responders. Additionally, the correlation between tumor markers (Day 14) and PET-CT SUVmax (Day 90) response was assessed. Only tumors with≥2.5 SUVmax were included in this analysis. Survival was measured from the date of radiographically documented disease progression and the date of death. Four patients still alive at the time of "data cut-off" (22/OCT/2018) were censored. Results: Median overall survival for the treated population is 8.8 months. Of the 41 implanted patients (51% female; 49% male) 56.1% were Responders (n = 23R) and 43.9% Non-Responders (n = 18 NR) as defined by the tumor markers preliminary evaluation post-implant. CEA levels were: Baseline, mean R 122.45; Day 14, mean R 59.311 vs. Baseline, mean NR 1610.31; Day 14, mean NR 1790.23 [p < 0.000041]. CA 19-9 levels were: Baseline, mean R 161.85; Day 14, mean R 64.69 vs. Baseline, mean NR 949.38889; Day 14, mean NR 1930.78 [p < 0.037901]. Additionally, a correlation was noted between CEA and/or CA19-9 responders and the SUVmax findings. Seventy-seven lesions were evaluated in 18/41 evaluable patients during the day 90 assessments. SUVmax ranged 2.5–34.9. Eleven (61%) of the 18 patients were SUVmax responders. Of these 11 SUVmax R patients - 8 were also tumor markers responders. We observed 3 [central (2)/partial (1)] necrotic lesions, 34 lesions with decreased tumor activity, 21 lesions with increased, and 19 lesions with disease stabilizations. Conclusion: The Macrobead studies provided evidence for survival benefit and enhanced quality of life for heavily pre-treated mCRC patients. The PET-CT SUVmax changes along with tumor markers response further support the anti-cancer effect of the RMBs. A Phase III study involving more than three hundred patients will soon be launched to further evaluate the potential of RMB as a new line of therapy for mCRC.

Introduction: Because cancer can be considered as a complex, biological-systems disease, we have explored a cell-based systems therapeutic approach based on this concept. We report ongoing human experience using mouse renal adenocarcinoma cells (RENCA) entrapped in agarose macrobeads (RMB) for late-stage mCRC therapy (Chin J Cancer Res 2018;30(1):72-83). Such cells undergo genomic changes that release multiple anti-neoplastic factors (> ten). One or more achieve 40% of their tumor-cell inhibitory effect via MEF-2 regulation. (Cancer Res. 2011;71(3): 716–724;725–735). Other pathways are currently under study with Lund University. Phase I and IIa RMB trials provided evidence of survival and quality of life benefit in treatment-resistant, late-stage mCRC patients (Cancer Growth Metastasis.2016;9:9–20). Here, we report the combined findings of the P I/IIa, along with new preliminary survival and other-benefit findings from a PIIb multi-site trial. Methods: Eighty-nine mCRC patients who failed all available cancer treatments underwent laparoscopic intraperitoneal implantation of RMBs up to 4 times in these open-label PI/IIa/IIb trials. The PIIb multi-site trial (40 patients), closed for enrollment, currently includes four in LTFU and one patient undergoing a fourth implantation. OS was the primary endpoint, with serial physical examinations, lab profiles and PET-CT imaging performed pre- and three months post-implantation to further evaluate safety/efficacy in all three trials. Survival was measured from time of study entry until death. The P I/IIA trials differed from IIb in that none of their patients had been treated with regorafenib or trifluridine/tipiracil. Results: Mean survival for 84/89 patients (all three trials) was 41 weeks [median survival=33 weeks; SD +/-37.5 wks]. For all patients in Phase IIb only (n = 36/41), preliminary analysis indicates mean survival of 34 weeks [median, 29.5 wks; SD +/-24 wks]. For patients who received regorafenib and/or trifluridine/tipiracil prior to RMBs in this trial (n = 12/13), mean survival was 26.5 weeks [median survival=21wks; SD+/-23 wks]. For the patients not receiving either (n = 23/36), mean survival was 37wks [median survival=35wks; SD+/-25 wks]. In all three trials, the ancillary testing described in Methods indicated that there were responders (R), as well as non-responders (NR) to the RMB. For the combined PI/IIa data, two groups of patients: R (n = 25) and NR (n = 9) were defined by their LDH values at days 30 and 60 after first implantation (D30, mean R value 305.92+/-284.76 vs. NR, 649.33+/- 363.60; p < 0.0070), D60 (R, 333.88+/-445.89 vs. NR, 1278.50+/- 761.9; p < 0.0001). These correlated with CEA and/or CA19-9 decreases (≥20%), and with the PET-CT SUVmax findings. Eighty-four FDG-positive mCRC lesions were detected in 26/34 patients at D90. Of the 25 LDH responders, 14 showed stable or decreased SUVmax values; seven an increase (4 non-evaluable) (doi:10.3252/pso.eu.19wcgic.2017). Parallel data from PIIb will be presented at WCGIC 2018. Conclusion: Taken together, the PI/IIa/IIb trial data are encouraging in indicating improved survival of late-stage mCRC patients with RMB treatment. The laboratory data (LDH levels, CEA and CA19-9) decreases, as well as PET-CT imaging (decreased SUVmax) support this. The data to date merit development of a Phase III randomized trial of RMB vs. standard therapy to further assess the effectiveness of the RMB in late-stage mCRC.

e13594 Background: This first in-human Phase I trial evaluates the treatment of AEC by the intraperitoneal implantation of RENCA MB. MB consist of two concentric agarose layers with RENCA in the inner layer. Of 150,000 cells originally in the inner layer, greater than 99% die. One sub-population of remaining cells has stem cell properties that form colonies and secrete factors to inhibit the proliferation of cancer cells outside the MB, in vitro or in vivo. Inhibition is neither species- nor tumor type–specific. Release of such inhibitory signals from cancer cells, in a proliferation-restrictive environment, may be a useful adjunct in human cancer. Primary endpoint: safety; secondary endpoint: efficacy. Methods: This open-label, IRB-approved IND trial enrolled 31 patients (pts) with AEC (rectum, pancreas, liver, colon, lung, prostate, ovarian). Informed consent obtained. Either 8 or 16 RENCA MB/kg implanted into the peritoneal cavity via laparoscopy. Serial physicals, lab cytokine profiles, and PET/CT or MRI were done pre- and post-implant to assess safety and efficacy. Results: 31 pts (15M: 16F) implanted with RENCA MB. Mean age 59. Mean number of MB implanted 809 (range 415-1520). 6/31 pts had >1 implant. MB were well tolerated with fatigue and anorexia lasting days to 3 wks. Less frequent adverse events (AE), not necessarily related to MB, were abdominal pain, constipation, pyrexia, nausea, vomiting, dyspnea, localized fluid collections around MB, ascites, abdominal distension, peripheral edema. Preliminary indicators of potential efficacy included decreases in tumor markers, disease stabilization, improvement in pain and quality of life (EORTC scale). 27/31 pts have died since 2005. 4 ongoing pts. Mean number of days from implant to death was 164 (range 13-607). None of the deaths were related to RENCA MB or to their implantation. Conclusions: Safety and toleration were demonstrated in 31 implanted pts. Most pts had a positive therapeutic effect. Serious AE and deaths were related to disease progression and complications from the underlying carcinomas.A phase II trial in pts with refractory pancreas and colorectal cancers is ongoing.

BACKGROUND:We investigated the effectiveness of implanted macrobeads containing porcine islets as long-term therapy for type I diabetes mellitus in Biobreeding/Worcester (BB/Wor) rats, an animal model of spontaneous type I human diabetes. End points included acute control of glucose, weight gain, survival time, and the renal changes associated with diabetes.MATERIALS AND METHODS:Eighteen chronic spontaneously diabetic BB/Wor rats were each implanted with 56-150 porcine islet macrobeads secreting 1.3-5.2 U of insulin/24 hr in culture medium at 37 degrees C. Their clinical courses and selective histological observations were compared with those of animals maintained on Linplant insulin-release implants (6 rats) or protamine zinc insulin alone (10 rats).RESULTS:The rats that underwent porcine islet macrobead implantation (PIMI) survived for a mean of 171 days (range, 79-288) after implantation without exogenous insulin, immunosuppressive treatment, or lactated Ringer's therapy. All appeared healthy and maintained their body weights (mean 356+/-21 g) throughout this period, even though their nonfasting blood glucose levels fluctuated significantly, with the mean for the group being 245+/-102 mg/dl (range, 157-320 mg/dl). There was mild glucosuria in some animals. In comparison, the 10 BB/Wor rats maintained on exogenous protamine zinc insulin had a mean survival time of 53 days (range, 10-217), a "last entry" mean body weight of 283+/-23 g, and a mean nonfasting glucose level of 340+/-90 mg/dl. The six Linplant implant animals had a mean survival time of 164 days (range, 1-264 days), a "last entry" mean body weight of 374+/-21 g, and a mean nonfasting glucose level of 189+/-91 mg/dl (range, 135-219). Episodes of ketonuria, abrupt loss of body weight, dehydration, and symptomatic hypoglycemia were more common in both these groups than in the PIMI animals. Glucose tolerance tests comparing diabetic animals treated with porcine islet macrobead implants, exogenous insulin-treated diabetic BB/Wor rats, and normal nondiabetic Wistar-Furth rats showed that the responses of those with the macrobead implants were similar to those of the normal rats, while the exogenous insulin-treated diabetic BB/Wor rats had the expected abnormal responses. Light microscopic examination of the PIMI and Linplant animals' kidney sections appeared normal, whereas those of the exogenous insulin-injected BB rats showed moderate focal tubular atrophy and an increased mesangial matrix. Macrobeads retrieved from the peritoneal cavity at necropsy were found to secrete insulin, C-peptide, and glucagon, indicating that they were still functional after 199 or more days in the peritoneal cavity.CONCLUSIONS:Our results indicate that macrobeads containing porcine islets implanted intraperitoneally in natural insulin-dependent diabetic BB/Wor rats are capable of normalizing glucose control, permitting a normal life span, and preventing the renal changes normally associated with diabetes. Therefore, further short- and long-term studies of porcine islet macrobead implantation in chemically induced and naturally occurring diabetes in rodents, as well as larger animals including dogs, monkeys and possibly humans, are merited.

Several obstacles have hindered the successful transplantation of islets of Langerhans to human patients in efforts to cure type I diabetes mellitus. One problem is the necessity for short- and long-term storage of islets after isolation and before transplantation. Current long-term storage methods, such as incubation in a physiological medium and cryopreservation, are suboptimal, resulting in significant loss of viable islet mass or function. Better storage methods are needed, In this study we examined the long-term storage of rat islets in macrobeads composed of agarose and collagen, Islets isolated from Wistar-Furth rats were placed into macrobeads (1000 islets/macrobead) and maintained in culture for periods of up to 189 days at 37 degrees C. Insulin released from the cultured macrobeads remained constant for periods of at least 154 days. In one group, insulin release was 1050 mU/24 hr/4 beads on day 3 and 1040 mU/24hr/4 beads on day 154. In another group, insulin release was 1305 mU/24 hr/5 beads on day 17 and 1580 mU/24 hr/5 beads on day 112. Xenotransplantation of Wistar Furth islet macrobeads, stored for 10 to 112 days at 37 degrees C, into 42 B6AF/1 mice with streptozotocin-induced diabetes resulted in a return to euglycemia in the recipients within 24 hr. Thereafter, euglycemia was maintained for more than 100 days in 32/42 of the recipients, and removal of the macrobeads caused a return to hyperglycemia within 48 hr in all animals. In addition, a group of 7 mice receiving macrobeads containing 1000 islets stored for 84 days had normal glucose tolerance tests (compared with those of 7 nontreated, nontransplanted mice with streptozotocin-induced diabetes and 7 normal mice), demonstrating that the islets in the macrobeads were functioning as they would in an intact pancreas. Finally, 5 macrobeads transplanted after initial storage of 112 days, removed from the first recipient after 100 days or more, stored again for 4 days in vitro, and retransplanted into 5 other diabetic mice also restored and maintained euglycemia for at least 45 days. Our results indicate that collagen-agarose macrobeads are capable of preserving rat pancreatic islets for extended periods without loss of in vitro insulin release capability or ability to achieve and maintain euglycemia in vivo, As such they should be useful for human islet transplantation efforts.

A Phase I trial of intravenous bromodeoxyuridine (BUdR) and conventional fractionated radiation therapy was performed in 14 patients with glioblastoma multiforme and 7 patients with other poorly radioresponsive tumors. The BUdR was given as a constant intravenous infusion for 12 hr/day for up to 14 days. Thirteen patients received a second 14 day infusion following a 10 to 14 day interruption for bone marrow recovery. Local toxicity (within the radiation field) was minor, with 7 of the 21 patients requiring a brief treatment break for moist skin desquamation. There was no significant CNS toxicity noted clinically nor by autopsy examination. Additionally, no significant enhancement of radiation injury was noted to bowel or liver. However, one patient treated for multiple pulmonary metastases experienced a clinical and radiographic pattern consistent with radiation pneumonitis. Dose-dependent systemic toxicity occurred in bone marrow and skin. Moderate myelosuppression, especially thrombocytopenia, was found following a 14 day cycle of BUdR at and above 650 mg/m2/12 hr infusion. Approximately one-third of patients developed a maculo-papular erythematous rash to the scalp, neck and upper chest. In two patients, the rash became generalized with evidence of epidermolysis on skin biopsy. Pharmacology studies revealed steady-state arterial plasma levels of 2 X 10(-6) M/1 during the 12 hr infusion of 650 to 700 mg/m2. Radiosensitization was measured by a change in the D0 of radiation survival curves of human bone marrow CFUc prior to and following the 14 day infusion in 4 patients. A trend of increasing radiosensitization was noted in most patients as the infusion rate of BUdR was increased from 500 to 870 mg/m2/12 hr. We conclude that the maximum tolerable dose of BUdR is 650 to 700 mg/m2/12 hrs when given as a 2 week intermittent intravenous infusion. Local toxicity is acceptable. The major systemic toxicities are myelosuppression and a maculopapular skin rash.

The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the National Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken from 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000-1500 mg) of BUdR were infused for 12 hours every 24 hours for up to 14 consecutive days. Cell survival was determined by colony formation of CFUc in soft agar suspension. X ray survival curves were generated over a dose range of 0-300 rad and the slopes of the survival curves (DO) before and after BUdR infusion were compared. Radiation enhancement ratios (ER) (DO pre-BUdR/DO post-BUdR) ranged from 1.0-2.2 and appeared to be BUdR dose dependent. Above 650 mg/m2, the radiation ER was greater than or equal to 1.5. Dose dependent systemic toxicity to bone marrow and skin was also observed with intermittent intravenous infusions of BUdR. From our study, it appears that an intravenous dose of less than 700 mg/m2/12 hours is well tolerated and may result in radiosensitization of CFUc in man.

An in vitro microcytotoxicity assay was utilized to determine the sensitivity of 58 cultured human malignant gliomas to the chemotherapy agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Of 58 such tumors, 42 (72%) showed a statistically significant cytotoxic response to BCNU in this assay. For those responding tumor lines, the cytotoxic index ranged from 0.25 to 0.76, with most clustered at the 0.40 level. To determine the therapeutic predictive relevance of such microcytotoxicity testing, the clinical course of patients receiving postoperative radiation therapy plus two or more doses of nitrosourea chemotherapy, as well as two or more computerized tomographic scans, was evaluated. In the 14 patients meeting all these criteria, tumor size increased in all five patients whose tumors did not respond to BCNU in the microcytotoxicity test. Six of the nine patients whose tumors in culture showed significant sensitivity to BCNU in vitro showed a clear decrease in tumor size over periods ranging from 17 to 48 months. Tumors in two patients increased in size, and one remained unchanged over the interval studied. These data support the concept that in vitro microcytotoxicity testing can be predictive of clinical response. Further study of this correlation seems warranted.

Chiro, G. Di; DeLaPaz, R.; Smith, B.; Kornblith, P.; Sokoloff, L.; Brooks, R.; Blasberg, R.; Cummins, C.; Kessler, R.; Wolf, A.; Fowler, J.; London, W.; Sever, J. Author Information

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A pronounced incorporation of 3H nucleotides into mitochondrial DNA in isolated suspensions of mitochondria has been demonstrated recently for a number of cell types. Although it is generally assumed that the multiplication of mitochondria is accompanied by replication of their DNA, pronounced incorporation of 3H-thymidine into the mitochondria of higher organisms in vivo has yet to be shown. In the present study, uptake has been measured of 3H-thymidine into neurons of mouse spinal ganglia, cockroach nerve cord ganglia and goldfish retina.