Novel unconventional radiotherapy techniques: Current status and future perspectives - Report from the 2nd international radiation oncology online seminar.

•Improvement of therapeutic ratio by novel unconventional radiotherapy approaches.•Immunomodulation using high-dose spatially fractionated radiotherapy.•Boosting radiation anti-tumor effects by adding an immune-mediated cell killing.

B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months.

Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Rejection in Gal knockout (GTKO) pigs due to elicited non-Gal antibody response required further genetic modifications of donor pigs and better control of the B-cell response to xenoantigens. We report significant prolongation of heterotopic alpha Galactosyl transferase "knock-out" and human CD46 transgenic (GTKO.hCD46Tg) pig cardiac xenografts survival in specific pathogen free baboons. Peritransplant B-cell depletion using 4 weekly doses of anti-CD20 antibody in the context of an established ATG, anti-CD154 and MMF-based immunosuppressive regimen prolonged GTKO.hCD46Tg graft survival for up to 236 days (n = 9, median survival 71 days and mean survival 94 days). B-cell depletion persisted for over 2 months, and elicited anti-non-Gal antibody production remained suppressed for the duration of graft follow-up. This result identifies a critical role for B cells in the mechanisms of elicited anti-non-Gal antibody and delayed xenograft rejection. Model-related morbidity due to variety of causes was seen in these experiments, suggesting that further therapeutic interventions, including candidate genetic modifications of donor pigs, may be necessary to reduce late morbidity in this model to a clinically manageable level.

Rapamycin promotes the enrichment of CD4(+)CD25(hi)FoxP3(+) T regulatory cells from naïve CD4(+) T cells of baboon that suppress antiporcine xenogenic response in vitro.

The CD4(+)CD25(+)FoxP3(+) regulatory T (Treg) cells play an important role in regulating the immune response. These Treg cells are present in peripheral blood and lymphoid organs and have a high potential for immunotherapy in clinics. Adoptive cell transfer therapy using CD4(+)CD25(+) cells has been shown to prevent autoimmune diseases and has also induced transplant tolerance in mice. Treg cells low frequency in peripheral blood will necessitate its ex vivo expansion to enable adaptive immunotherapy. Recently, it has been reported that rapamycin, an immunosuppressive agent, inhibits T-cell proliferation while selectively increasing the number of Treg cells. Based on this additional mode of action, rapamycin can be used to expand Treg cells for ex vivo cellular therapy in T-cell-mediated diseases and in transplantation. We have reported the ex vivo expansion of baboon Treg cells, using irradiated pig peripheral blood mononuclear cell (PBMC) and interleukin (IL)-2, and have demonstrated the suppression of autologus CD4(+)CD25(neg) T-cell proliferation in response to pig PBMCs. In the present study, we have expanded baboon CD4(+) T cells in the presence or absence of rapamycin (0.1-10 nmol/L) using irradiated pig PBMCs and IL-2 to enrich the regulatory T cells. CD4(+)CD25(+)FoxP3(+) Treg cells were increased up to 2 times in the presence of rapamycin versus without rapamycin in vitro. However, a higher dose of rapamycin (> or = 10 nmol/L) considerably decreases the number of Treg cells. Furthermore, purified CD4(+)CD25(+) Treg cells enriched from CD4(+) cells in the presence of rapamycin were able to suppress the baboon anti-porcine xenogeneic immune responses in vitro up to 93% at a 1:1 ratio (Treg cells:T effector cells) and suppression ability exists even at a 1:256 ratio, whereas freshly isolated natural Treg cells suppress only 70% at 1:1 and lose their suppressive ability (>50%) at 1:16. Our results demonstrate that the addition of rapamycin to the culture enriches the Treg phenotype and induces functional regulatory T cells. This method may allow the production of large numbers of regulatory cells for the preclinical testing of Treg cell therapy in a non-human primate model.

Spatially fractionated (GRID) radiation therapy using proton pencil beam scanning (PBS): Feasibility study and clinical implementation.

PURPOSE: GRID therapy is an effective treatment for bulky tumors. Linear accelerator (Linac)-produced photon beams collimated through blocks or multileaf collimators (MLCs) are the most common methods used to deliver this therapy. Utilizing the newest proton delivery method of pencil beam scanning (PBS) can further improve the efficacy of GRID therapy. In this study, we developed a method of delivering GRID therapy using proton PBS, evaluated the dosimetry of this novel technique and applied this method in two clinical cases. MATERIALS/METHODS: In the feasibility study phase, a single PBS proton beam was optimized to heterogeneously irradiate a shallow 20 × 20 × 12 cm3 target volume centered at a 6 cm depth in a water phantom. The beam was constrained to have an identical spot pattern in all layers, creating a "beamlet" at each spot position. Another GRID treatment using PBS was also performed on a deep 15 × 15 × 8 cm3 target volume centered at a 14 cm depth in a water phantom. Dosimetric parameters of both PBS dose distributions were compared with typical photon GRID dose distributions. In the next phase, four patients have been treated at our center with this proton GRID technique. The planning, dosimetry, and measurements for two representative patients are reported. RESULTS: For the shallow phantom target, the depth-dose curve of the PBS plan was uniform within the target (variation < 5%) and dropped quickly beyond the target (50% at 12.9 cm and 0.5% at 14 cm). The lateral profiles of the PBS plan were comparable to those of photon GRID in terms of valley-to-peak ratios. For the deep phantom target, the PBS plan provided smaller valley-to-peak ratios than the photon GRID technique. Pretreatment dose verification QA showed close agreement between the measurements and the plan (pass rate > 95% with a gamma index criterion of 3%/3 mm). Patients tolerated the treatment well without significant skin toxicity (radiation dermatitis grade ≤ 1). CONCLUSIONS: Proton GRID therapy using a PBS delivery method was successfully developed and implemented clinically. Proton GRID therapy offers many advantages over photon GRID techniques. The use of protons provides a more uniform beamlet dose within the tumor and spares normal tissues located beyond the tumor. This new PBS method will also reduce the dose to proximal organs when treating a deep-seated tumor.

Severe acute enteritis in a multiple myeloma patient receiving bortezomib and spinal radiotherapy: case report.

Proteasome inhibitors have been reported to enhance radiosensitivity in vitro. A case of potential clinical interaction between bortezomib, a proteasome inhibitor, and spine radiation is reported. A woman undergoing palliative radiotherapy to the T12 -S2 spine with concurrent bortezomib developed unexpectedly severe, acute radiation enteritis requiring hospital admission. Clinicians are advised to consider the potential for interactions of bortezomib with radiotherapy when the two agents are used simultaneously in the clinic.

Establishment of fully xenogeneic (mouse-->rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells.

BACKGROUND: Xenotransplantation is a potential solution to the critical shortage of transplantable organs. However, conventional immunosuppressive agents do not control the vigorous cellular and humoral rejection across species disparities. The induction of donor specific tolerance via bone marrow chimerism may be a method to avoid xenograft rejection. In xenogeneic chimeras, T cell repertoire selection plays an important role in the induction of tolerance. Until now a model of mouse-->rat multilineage chimerism has not been reported. This study reports the establishment of fully xenogeneic mouse-->rat multilineage chimeras and evaluates the role of T cell development and repertoire selection in tolerance induction in a xenogeneic environment. METHODS: Recipient rats were irradiated at a dose of total body irradiation ranging between 800-1100 cGy and injected with 120-300x10(6) donor mouse bone marrow cells. Chimeras were typed for engraftment at 4 weeks and then monthly thereafter. T cell repertoire was evaluated in chimeras using two-color flow cytometry and monoclonal antibodies directed against the variable portion of the beta chain of the T cell receptor. RESULTS: Fully xenogeneic multilineage bone marrow chimerism was produced in a mouse-->rat model by using ablative radiation and a high dose of donor cells. Mouse T cells develop in a phenotypically normal fashion in chimeric rats and the host rat is capable of deleting T cells that are reactive to the donor mouse strain. CONCLUSION: Long-term multilineage bone marrow chimerism can be produced in a mouse-->rat bone marrow transplant model. Mouse T cells develop in a phenotypically normal fashion and negative selection of specific T cell receptor-Vbeta occurs in a xenogeneic environment in a predictable fashion paralleling that for syngeneic or allogeneic transplantation.

Long-term survival of cardiac xenografts in fully xenogeneic (mouse --> rat) bone marrow chimeras.

BACKGROUND: The shortage of human hearts remains a major barrier to the efficacy of heart transplantation for the treatment of end-stage heart disease. One potential solution to the supply problem would be the use of hearts from nonhuman donors (xenografts). We have established a model of mouse to rat xenogeneic bone marrow chimerism, and in this study we have hypothesized that such chimeric rats will accept both donor and recipient specific heart grafts while rejecting third-party mouse and rat grafts. We also investigated humoral responses in naive and chimeric rats with and without donor murine cardiac grafts. METHODS: Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 10(6) donor B10.BR mouse bone marrow cells intravenously. Peripheral blood of surviving rats (n = 18) was typed at 4 weeks and then monthly thereafter. Donor and recipient specific and third-party heterotopic heart transplantations were performed at 6 to 8 weeks after reconstitution with bone marrow. RESULTS: Multilineage bone marrow chimerism was produced in all experimental animals with complete replacement of recipient marrow by donor cells. Murine donor and rat recipient strain hearts transplanted in chimeric rats survived indefinitely. Third-party rat and mouse hearts were rejected, though at a slower rate than bone marrow matched naive controls. High levels of antimouse antibodies were detected in rats with rejected hearts. These antibodies were absent in chimeric animals with long-term surviving heart grafts. CONCLUSIONS: Long-term multilineage bone marrow chimerism can be produced in a mouse --> rat bone marrow transplant model. Long-term survival of donor specific and recipient specific vascularized cardiac grafts can be produced in these chimeric animals. These animals are clinically normal but show signs of subclinical immunosuppression regimen as they reject third-party hearts later than naive animals. Our results suggest that antibodies also play a significant role in concordant xenograft rejection, and induction of bone marrow chimerism can overcome this barrier.

A novel technique for post-mastectomy breast irradiation utilising non-coplanar intensity-modulated radiation therapy.

The aim of this study was to investigate if non-coplanar intensity-modulated radiation therapy (IMRT) in the post-mastectomy setting can reduce the dose to normal structures and improve target coverage. We compared this IMRT technique with a standard partial wide tangential (PWT) plan and a five-field (5F) photon-electron plan. 10 patients who underwent left-sided mastectomy were planned to 50.4 Gy using either (1) PWT to cover the internal mammary (IM) nodes and supraclavicular fields, (2) 5F comprising standard tangents, supraclavicular fields and an electron field for the IM nodes or (3) IMRT. The planning target volume (PTV) included the left chest wall, supraclavicular, axillary and IM lymph nodes. No beams were directed at the right lung, right breast or heart. Mean dose-volume histograms were constructed by combining the dose-volume histogram data from all 10 patients. The mean PTV to receive 95% of the dose (V95%) was improved with the IMRT plan to 94.2% from 91.4% (p = 0.04) with the PWT plan and from 87.7% (p = 0.012) with the 5F plan. The mean V110% of the PTV was improved to 3.6% for the IMRT plan from 16.8% (p = 0.038) for the PWT plan and from 51.8% (p = 0.001) for the 5F plan. The mean fraction volume receiving 30 Gy (v30Gy) of the heart was improved with the IMRT plan to 2.3% from 7.5% (p = 0.01) for the PWT plan and 4.9% (p = 0.02) for the 5F plan. In conclusion, non-coplanar IMRT results in improved coverage of the PTV and a lower heart dose when compared with a 5F or PWT plan.

Left ventricular pressure measurement by telemetry is an effective means to evaluate transplanted heart function in experimental heterotopic cardiac xenotransplantation.

Evaluation of the function of heterotopic cardiac transplants has traditionally been accomplished by either manual palpation or serial biopsies. Both methods have drawbacks. Palpation can be difficult to differentiate a pulse from the graft versus a transmitted pulse from the native aorta. Serial biopsies, though accurate, require multiple laparotomies, leading to increased morbidity and possibly mortality rates. In this study we used an advanced telemetry system, consisting of an intra-abdominal implant, that was capable of continuously monitoring simultaneously several parameters of the transplanted heart and the status of the recipient. In a large animal model of heterotopic cardiac xenotransplantation (pig donor to baboon recipient), we implanted the device in 12 animals: 8 with and 4 without immunosuppression. We monitored and continuously recorded the left ventricular pressure (both peak-systolic and end-diastolic [LVEDP]), heart rate, and the electrocardiogram pattern of the transplanted heart as well as the temperature of the recipient. The left ventricular pressure proved to be the most valuable parameter to assess graft heart function. In the 4 nonimmunosuppressed cases, grafts were rejected acutely. In these cases, the end-diastolic pressure increased sharply and the heart stopped contracting when the difference between the systolic and the diastolic pressure decreased to <10 mm Hg. The earliest reproducible sign of rejection was an increased LVEDP. Among long-term survivors, the increase in diastolic pressure was gradual, indicating progressive thickening of the myocardium and decreased compliance of the ventricle. Six of 8 immunosuppressed animals died of other complications before rejecting the transplanted heart. The telemetry was also helpful to indicate early onset of fever in the recipients, thus allowing us to intervene early and prevent potentially lethal septic complications. Continuous monitoring of several parameters via telemetry allowed detection of changes associated with rejection as well as other complications at an early stage, allowing prompt intervention, treatment, and possibly reversal of rejection.

Surgical and nonsurgical complications of a pig to baboon heterotopic heart transplantation model.

A modified immunosuppressive regimen, developed at the National Institutes of Health, has been employed in a large animal model of heterotopic cardiac xenotransplantation. Graft survival has been prolonged, but despite this, our recipients have succumbed to various surgical or nonsurgical complications. Herein, we have described different complications and management strategies. The most common complication was hypercoagulability (HC) after transplantation, causing thrombosis of both small and large vasculature, ultimately leading to graft loss. While managing this complication we discovered that there was a delicate balance between HC and consumptive coagulopathy (CC). CC encountered in some recipient baboons was not able to be reversed by stopping anticoagulation and administering multiple blood transfusions. Some complications had iatrogenic components. To monitor the animals, a solid state left ventricular telemetry probe was placed directly into the transplanted heart via the apex. Induction of hypocoagulable states by continuous heparin infusion led to uncontrollable intra-abdominal bleeding in 1 baboon from this apical site. This occurrence necessitated securing the probe more tightly with multiple purse strings and 4-quadrant pledgeted stay sutures. One instance of cardiac rupture originated from a lateral wall infarction site. Earlier studies have shown infections to be uniformly fatal in this transplant model. However, owing to the telemetry placement, infections were identified early by temperature spikes that were treated promptly with antibiotics. We had several cases of wound dehiscence due to recipients disrupting the suture line. These complications were promptly resolved by either re-approximating the wound or finding distractions for the baboon. A few of the most common problems we faced in our earlier experiments were related to the jacket, tether, and infusion pumps. It was difficult to keep the jackets on some baboons and the tether had to be modified several times before we assured long-term success. Infusion catheter replacement resulted in transplant heart venous obstruction and thrombosis from a right common femoral venous line. Homeostatic perturbations such as HC and CC and baboon-induced wound complications comprised most complications. Major bleeding and death due to telemetry implantation and infarct rupture occurred in 2 baboons. Despite the variety of complications, we achieved significant graft prolongation in this model.

High-dose preoperative radiation and full-thickness local excision. A new option for patients with select cancers of the rectum.

Faced with the responsibility of treating patients with invasive distal rectal cancer who were medically unacceptable for the indicated radical surgery, a prospective study was initiated in which high dose preoperative radiation and full-thickness local excision were used. High dose preoperative radiation permitted full-thickness local excision of select cancers, which, by conventional standards, otherwise would have required radical surgery and permanent colostomy. Feasibility was measured on the basis of safety of the technique, control of the cancer, and the quality of anal sphincter function expected. Patients were selected initially because of their predicted inability to tolerate radical surgery, but indications were broadened to include those whose tumors had completely disappeared after irradiation. From 1984 to 1988, 20 patients underwent 21 operative procedures for cancers located between 0 and 7 cm from the anorectal ring. This report is concerned with the 14 patients of this group who were observed for a minimum of 24 months. High-dose preoperative radiation was administered for a total dose of 4500 cGy. Excision and repair were performed 4 to 6 weeks after completion of radiation therapy. Full-thickness disc or hemicircumferential excision was accomplished by transanal, transsphincteric, and transsacral techniques, which included, in several instances, excision of the sphincter mechanism and perineal body, and/or the vaginal wall. Full-thickness local excision after high-dose radiation therapy for rectal cancers has never been reported. Follow-up observation ranged from 24 to 48 months with a median of 31 months. Rectal reservoir function and sphincter control were good in 13 patients. Local recurrence developed in three patients (21 percent), two of whom had postradiation therapy B2 mucinous cancers. Three-year actuarial rate of local recurrence is 23 percent. One (7 percent) patient died of recurrent disease. Actuarial Kaplan-Meier survival at 3 years is 61 percent. Based on the results of this small, select patient group, high-dose radiation therapy followed by full-thickness local excision appears to be a reasonable option for patients who cannot tolerate radical surgery. This bimodal approach also may serve as an option for those who are good medical risks, but for whom sphincter preservation is at stake, and to whom radical surgery offers limited benefits.