Multiple antibodies targeting tumor-specific mutations redirect immune cells to inhibit tumor growth and increase survival in experimental animal models.

BACKGROUND: T cell therapy for cancer involves genetic introduction of a target-binding feature into autologous T cells, ex vivo expansion and single large bolus administration back to the patient. These reprogrammed T cells can be highly effective in killing cells, but tumor heterogeneity results in regrowth of cells that do not sufficiently express the single antigen being targeted. We describe a cell-based therapy that simultaneously targets multiple tumor-specific antigens. METHODS: High-affinity polyclonal rabbit antibodies were generated against nine different surface-related tumor-specific mutations on B16F10 cells. Unsorted splenic effector cells from syngeneic mice were incubated with a cocktail of the nine anti-B16F10 antibodies. These 'armed' effector cells were used to treat mice previously inoculated with B16F10 melanoma cells. RESULTS: The cocktail of nine antibodies resulted in dense homogeneous binding to histological sections of B16F10 cells. Five treatments with the armed effector cells and PD1 inhibition inhibited tumor growth and improved survival. Shortening the interval of the five treatments from every three days to every day increased survival. Arming effector cells with the four antibodies showing best binding to B16F10 cells even further increased survival. CONCLUSIONS: This study demonstrates that ex vivo arming a mixed population of immune effector cells with antibodies targeting multiple tumor-specific mutated proteins in conjunction with PD1 inhibition delayed tumor growth and prolonged survival in mice inoculated with an aggressive melanoma. A remarkably low total antibody dose of less than 5 µg was sufficient to accomplish tumor inhibition. Scaling up to clinical level may be feasible.

Hyperthermic enhancement of rhodamine 123 cytotoxicity in B16 mouse melanoma cells in vitro.

The effect of elevated temperature on cytotoxicity of rhodamine 123 (R123) was tested in vitro on B16 mouse melanoma cells. Simultaneous 1-h exposure to R123 and hyperthermia (43 degrees C for 1 h) resulted in marked enhancement of R123 cytotoxicity. Thermal enhancement of R123 cytotoxicity occurred at temperatures as low as 38 degrees C. Heat treatment (43 degrees C for 1 h) given immediately before or after R123 exposure (37 degrees C for 1 h) yielded no significant increase in cytotoxicity over that expected for strict additivity. The effects of heat on two mechanisms reported to be associated with R123 cytotoxicity were evaluated: (a) target inactivation by R123; and (b) R123 intracellular accumulation. Hyperthermia caused an increased rate of target inactivation by R123 and also caused an increased net intracellular accumulation of R123. This indicates that at least two mechanisms are responsible for the synergistic cytotoxicity of R123 and hyperthermia.

In vitro and in vivo cytotoxicity of rhodamine 123 combined with hyperthermia.

Because both Rhodamine 123 (R123) and hyperthermia have been shown to be cytotoxic, we examined their effect, independently and in combination, on five different human malignant cell lines in vitro and on cultured melanoma cells grown intradermally in nude mice. The cell lines examined include two human melanomas, UCLA-SO-M14 and UCLA-SO-M21, the colon cancer cell line HT29, the human lung cancer cell line P3, and the human breast cancer cell line B231. R123 and hyperthermia, when used in combination, were found to be cytotoxic for these five different human malignant cell lines in vitro. The two agents together appear to enhance the cytotoxic effect of each alone, as documented by synergistic ratios ranging from 2.31 to 45 for the different cell lines. In the "nude" mouse model, animals were treated with a combination of R123 and hyperthermia (43 degrees C for 90 min). A statistically significant (P = 0.04) decrease in tumor growth rate was observed when compared with the rate of tumor growth in untreated animals. The results suggest a potential role for R123 in combination with hyperthermia in the treatment of malignant cells.

Tamoxifen and cardiac risk factors in healthy women: Suggestion of an anti-inflammatory effect.

-Tamoxifen reduces the incidence of breast cancer in women at risk for that disease. Because heart disease is the leading cause of death in women and because tamoxifen is also associated with venous thrombosis, an improved understanding of the association of tamoxifen with cardiovascular disease risk factors is required. In 111 healthy women at a single center, who were participating in a randomized double-blind breast cancer prevention trial, the 6-month effects of oral tamoxifen (20 mg/d) compared with placebo on factors related to inflammation, hemostasis, and lipids were studied. Tamoxifen was associated with reductions of 26% in median C-reactive protein, 22% in median fibrinogen, and 9% in cholesterol (all P:<0.01 compared with placebo). There were no differences in treatment effects on factor VII coagulant activity, fragment 1-2, and triglycerides. In secondary analyses, the effect of tamoxifen on C-reactive protein was larger in postmenopausal women and in women with higher waist-to-hip ratios. The effect on fibrinogen was larger in women with higher baseline cholesterol. Tamoxifen demonstrated effects on inflammatory markers that were consistent with reduced cardiovascular risk. These findings are in contrast to recent reports of increased C-reactive protein associated with postmenopausal estrogen. The potential for beneficial cardiovascular effects of tamoxifen in healthy women is suggested.

Heterogeneity of heat response in murine, canine and human tumors: influence on predictive assays.

The heterogeneity of response to hyperthermia of cells taken from different regions of tumors was tested in a model tumor system (RIF-1) in the mouse and in specimens from spontaneous tumors taken from dogs and humans at the time of surgical resection. Cell survival was assayed by clonogenic survival in the murine tumor and by dansyl lysine staining in tumors from all three species. Using survival as an endpoint, it was found that the extent of heterogeneity depended on the temperature to which the tumor was heated and the duration of exposure. By increasing either of these factors, the coefficient of variation was increased. The large heterogeneity seen after in vivo heating could not be explained entirely by inhomogeneous heating within the tumor as evidenced by temperature mapping. It is concluded that other microenvironmental factors such as blood flow, pH, O2, and nutrient supply may cause variations in the heat response of the tumor cells in vivo. Little, if any, evidence of cellular heterogeneity was evident for all three species when comparisons were made between samples of 100-200 mg. The canine and human tumors were considerably more heat resistant when dansyl lysine was used as an endpoint. In the RIF-1 tumors, heterogeneity of heat response was greater after in vitro heating than after in vivo heating when small biopsy samples (10-20 mg) were taken, suggesting that some cellular heterogeneity was present.

Clinical utility of immunoscintigraphy in managing ovarian cancer.

Ovarian cancer spreads to multiple areas of the peritoneal cavity early in the course of the disease. Multiple small foci of tumor are not identified readily with standard preoperative staging procedures. A review of an immunoscintigraphy study of more than 100 women preoperatively staged with 111In-labeled B72.3 (CYT-103) reveals that this technique has a low potential for serious side effects and it can identify miliary spread of ovarian cancer and extra-abdominal metastases. It has the potential to contribute favorably to patient management by detecting occult lesions, may define the extent of tumor as well or better than computerized tomography and may impact on surgical decision-making.

Sentinel lymph node localization in early breast cancer.

METHODS: Thirty-two patients with clinical node-negative breast cancer underwent sentinel node localization study as part of a National Cancer Institute-sponsored multicenter trial. Anatomical and histopathologic characteristics of sentinel lymph node (SLN) and a kinetic analysis of nodal uptake were studied. Patients were injected with 1 mCi/4 ml unfiltered 99mTc-sulfur colloid in four divided doses around the palpable lesion or immediately adjacent to the excision cavity if prior biopsy was performed. SLN biopsy was performed 1.5-6 hr (mean = 3 hr) postinjection. Intraoperative localization was performed using a gamma probe. All patients underwent complete axillary dissection. RESULTS: SLN was identified in 30 of 32 (94%) patients. There were no false-negative SLN biopsies. CONCLUSION: This study supports the clinical validity of SLN biopsy in breast cancer and confirms that, unlike the blue dye technique, the learning curve with unfiltered 99mTc-sulfur colloid and the gamma detection probe is short, and SLN localization is achievable in over 90% of cases by surgeons with modest experience. The use of unfiltered 99mTc-sulfur colloid (larger particle size) with larger injected volume permits effective localization of SLNs.

Composite tissue transfer in limb-salvage surgery.

After extensive resection due to extremity sarcoma, the inability to cover the defect for satisfactory healing and limb function has been an indication for amputation rather than limb salvage. We report herein our experience with seven limb-salvage cases in which we closed difficult and complex defects with composite tissue transfers utilizing microvascular techniques. Free-flap transfers were used to cover soft-tissue defects after extensive resection of primary and locally recurrent tumor and to manage radiation-induced complications. The grafts healed well when infected irradiated tissue was covered, and the grafts tolerated postoperative irradiation. Composite tissue transfer also provided soft-tissue coverage around distal joints that would not have been adequately protected with a skin graft. Complications were minimal, and all patients maintained good extremity function. No patient who underwent composite tissue transfer has had a local recurrence. A free-flap composite tissue transfer can extend the indications for limb-salvage surgery and offers an alternative to amputation in selected patients.

Clinical immunoscintigraphy of recurrent ovarian cancer with indium 111-labeled B72.3 monoclonal antibody.

OBJECTIVE: To prospectively evaluate the ability for immunoscintigraphy with monoclonal antibody CYT-103 labeled with indium 111 to detect tumor presence in 15 patients with ovarian cancer undergoing second-look surgery. DESIGN: Prospective, open-label, nonrandomized trial. SETTING: Hospital-based nuclear medicine facility and operating room. STUDY PARTICIPANTS: Patients with previous ovarian cancer scheduled for second-look surgery. MAIN OUTCOME MEASURE: Correctness of prediction of immunoscintigraphy for presence or absence of ovarian cancer compared with serum CA 125 titer and computed tomography. RESULTS: Immunoscintigraphy, computed tomography, and serum CA 125 titer had respective sensitivities of 92%, 42%, and 42%; specificities of 67%, 100%, and 100%; accuracies of 87%, 53%, and 53%; and diagnostic values of 59%, 42%, and 42%. The full regional extent of recurrent tumor was correctly detected in 45% of patients by immunoscintigraphy and in none of the patients by computed tomography. Immunoscintigraphy detected miliary tumor in two of four patients and computed tomography, as expected, was unable to detect miliary disease. CONCLUSIONS: Recurrent ovarian cancer often presents as multiple small lesions throughout the abdominal cavity. In this subset of patients, immunoscintigraphy may be particularly well suited for detection of the presence of recurrent tumor.

Intraoperative autotransfusion in hepatic resection for malignancy. Is it safe?

To evaluate whether intraoperative autologous transfusion increases the risk of hematogenous dissemination of tumor we reviewed the risk of lung metastasis as well as disease-free and long-term survival of patients who underwent resection of malignant hepatic neoplasms with this technique. A retrospective review of patients undergoing liver resection for malignant disease revealed 39 patients in whom intraoperative autologous transfusion was used. The 2-year actuarial survival in the patients in this series, as calculated with the Kaplan-Meier method, was predicted to be 75%. Two-year actuarial disease-free survival was predicted to be 28%, and the risk of developing lung metastasis at 3 years was estimated to be 40%. The predicted overall survival and risk of recurrence in this series compare favorably with published data for patients in whom intraoperative autologous transfusion was not used.

Minimal-access surgery for staging of malignant melanoma.

OBJECTIVE: To develop a simple, minimally invasive technique of determining whether regional node metastasis has occurred in patients with melanoma. SETTING: Teaching hospital tertiary care and private practice settings. PATIENTS: Between February 1993 and October 1994, 121 patients with invasive malignant melanoma and clinically negative lymph nodes were enrolled in this clinical trial. DESIGN: Consecutive sample clinical trial. Within 24 hours prior to lymph node resection, a radioactive tracer was injected into the dermis around the site of the primary melanoma. Forty-four patients also had blue dye injected immediately prior to surgical resection. Measurement of radioactivity in the lymph nodes and surgical localization were made using a handheld gamma detector. Radiolabeled nodes were selectively removed with the least dissection possible. In patients with pathologically positive radiolabeled nodes, regional lymphadenectomy was performed. OUTCOME MEASURES: Successful identification of radiolabeled sentinel lymph nodes, correlation of radiolabeling with injection of blue dye, and regional node recurrence rate. RESULTS: Surgeons successfully resected the radiolabeled sentinel lymph nodes in 118 (98%) of 121 patients. One hundred percent of blue-stained lymph nodes were successfully radiolabeled. Fifteen patients had pathologically positive sentinel lymph nodes. In 10 patients, the sentinel node was the only node with metastasis. Two systemic and one regional node recurrences occurred during a mean follow-up of 220 days. CONCLUSIONS: Selective gamma probe-guided resection of the radiolabeled sentinel lymph node is possible in over 95% of patients with melanoma. This technique offers a simple and reliable method of staging of regional lymph nodes in these patients without performing a regional lymphadenectomy.

Intraoperative ultrasound localization to guide surgical excision of nonpalpable breast carcinoma.

BACKGROUND: This report describes a technique of intraoperative tumor localization by ultrasound without the use of a needle or wire to guide the excision of nonpalpable breast cancers. The results of our experience with pathologic margin status are reviewed. STUDY DESIGN: From 1994 to 1998, 65 breast cancers in 62 patients with biopsy-proved nonpalpable breast cancer were excised using intraoperative ultrasound localization. The pathologic status of the margins from the initial surgical excision specimen and any further excisions, either at the first operation or later procedures, was recorded. The distance from the tumor to the closest margin of excision was also determined. RESULTS: The overall success in achieving pathologically negative excision margins at first operation was 97% (63 of 65 cancers). Three patients underwent a second operative procedure, two for positive margins and one for a margin less than 1 mm (second operation = 4.8% of patients). After completion of the first operative procedure, the mean distance to the closest margin of excision was 0.8 cm. CONCLUSIONS: Intraoperative ultrasound localization for excision of nonpalpable breast cancers is feasible and gives results, in terms of pathologic margins, that are comparable with those achieved by standard needle-wire-guided excisions.

Limitation in gamma probe localization of the sentinel node in breast cancer patients with large excisional biopsy.

BACKGROUND: Radiolocalization and selective biopsy of the sentinel node to correctly predict the status of remaining lymph nodes may provide an alternative to axillary dissection in selected breast cancer patients with clinically negative lymph nodes. STUDY DESIGN: In a nonrandomized, multicenter clinical trial, gamma probe localization for lymphatic mapping and sentinel node biopsy along with axillary dissection was performed on 75 patients with invasive breast cancer and clinically negative lymph nodes. The accuracy of the sentinel node biopsy to correctly predict the status of the remaining axillary lymph nodes was established through standard pathologic investigation. RESULTS: A sentinel node was identified in 70 of 75 patients with a technical success rate of 93%. Of these 70 patients, 21 (30%) had axillary nodal metastases identified pathologically. Four of these 21 (19%) had sentinel nodes negative for metastases. All 4 false-negative patients had prior excisional biopsies. The false-negative group had a larger mean maximal biopsy dimension than the true-positive group. Eleven of the 21 patients with axillary metastases had a diagnosis made by core needle biopsy with no false negatives. CONCLUSIONS: The accuracy of the sentinel node biopsy in correctly predicting the status of remaining axillary lymph nodes may be limited in patients with large excision before radiolocalization of the sentinel node. Our findings suggest that excisional biopsy should be avoided prior to lymphatic mapping for sentinel node biopsy.

Gamma-probe guided localization of lymph nodes.

The initial draining lymph node (Sentinel node) of a tumour may reflect the status of the tumours spread to the remaining lymphatic bed. The sentinel node, which has been reported to predict metastatic melanoma, has recently been localized by a new invasive technique [1]. The goal of our pre-clinical trial was to test a non-invasive technique to localize the sentinel node. Gamma-probe guided localization was used to identify and then surgically remove the first draining lymph node(s) in 16 inguinal lymphatic basins of eight cats. This method was found to be comparable to an invasive method using a blue dye. Gamma-probe localization has several potential advantages in that it can: (a) precisely locate on the surface of the skin the position of an underlying lymph node, (b) intraoperatively guide the surgeon to the lymph node during dissection, (c) verify that the correct node has been biopsied, (d) determine the possible presence of residual lymph nodes, (e) allow lymph nodes to be harvested through a small incision as opposed to raising a skin flap, and (f) be rapidly and easily performed.

Surgical resection and radiolocalization of the sentinel lymph node in breast cancer using a gamma probe.

We have recently reported on a technique of gamma probe localization of radiolabelled lymph nodes to identify the sentinel node in malignant melanoma. In order to determine whether this technique is applicable to assist in staging breast cancer, a pilot study was begun to address two questions: (i) can the sentinel lymph node draining a breast cancer be identified for selective resection; and (ii) is the sentinel lymph node predictive of the status of the entire axillary lymph nodes? One to four hours prior to axillary lymph node dissection, 22 consecutive patients had approximately 0.4 mCi of technetium sulfur colloid in 0.5 ml saline injected around the perimeter of the breast lesion. A hand-held gamma counter was used at surgery to locate the lymph node(s) receiving drainage from the breast. A sentinel lymph node was identified in 18 of 22 patients. Of these 18 patients, the sentinel lymph node was positive in seven of seven patients, with pathologically verified metastatic breast cancer to at least one lymph node. In three out of seven patients, the sentinel lymph node was the only lymph node with metastatic cancer. In this pilot study of breast cancer patients, we conclude that: (i) radiolocalization and selective resection of sentinel lymph nodes is possible; and (ii) the sentinel lymph node appears to predict correctly the status of the remaining axilla. These data justify a larger clinical trial to verify the value of this technique.

Gamma-probe-guided lymph node localization in malignant melanoma.

The initial draining lymph node (sentinel node) has been successfully localized using intraoperative vital dye mapping and reportedly is predictive of regional nodal metastases in Clinical- Stage 1 melanoma. In an animal model, we previously established the technique of gamma-probe-guided localization of the technetium-99 sulfur colloid labelled sentinel node and found its sensitivity equal to vital dye mapping. We now report our initial experience using gamma-probe-guided localization to identify and then surgically remove the first draining lymph node(s) in 10 malignant melanoma patients. Lymphoscintigraphy was used to confirm localization. We conclude that this technique: (a) reliably localizes the sentinel node draining the site of a primary melanoma, (b) allows the lymphatic bed to be checked intraoperatively verifying complete sentinel node biopsy, and (c) is relatively simple and can be performed under local anaesthesia.

A simplified technique to resect abnormal bony radiolocalizations using a gamma counter.

A simplified technique for localizing and verifying the correct biopsy site of lesions identified on a bone scan has been utilized. A hand-held gamma counter was used for localization of incision placement, determination of extent of bone to be resected, and verification that appropriate tissue was resected. This technique was used to guide biopsy of bony lesions in five patients and to guide resection of a pubic ramus chondrosarcoma. We conclude that intraoperative use of a gamma counter to guide biopsy of bony lesions minimizes surgery time, increases the confidence of obtaining correct tissue, and makes a frequently frustrating procedure very simple. In addition, the probe may assist with determining adequate margins at definitive resection of tumours which accumulate technetium-99m MDP.

Psychological adjustment in breast cancer: processes of emotional distress.

The process of psychological adjustment to breast cancer was examined at diagnosis and at 3- and 6-month follow-ups in a sample of 80 women with Stage I-Stage IV breast cancer. At diagnosis, symptoms of anxiety/depression were predicted by low dispositional optimism, and this path was partially mediated by use of emotion-focused disengagement coping. Younger age also was predictive of anxiety/depression symptoms at time of diagnosis, and this relationship was fully mediated by magnitude of intrusive thoughts. At 3 months, changes in anxiety/depression symptoms were predicted only by intrusive thoughts. At 6 months, low dispositional optimism reemerged as a significant predictor of changes in anxiety/depression and again was partially mediated by the use of emotion-focused disengagement coping. Independent effects for problem-focused engagement and disengagement and emotion-focused engagement coping were also found at 6 months. Implications of these data for psychosocial interventions with breast cancer patients are highlighted.

Transport and retention of colloidal tracers in regional lymphoscintigraphy in melanoma: influence on lymphatic mapping and sentinel node biopsy.

Sentinel node (SN) biopsy is a staging technique used to select patients for regional lymphadenectomy in melanoma. We compared the two most widely used radioactive tracers, 99mTc-colloidal albumin (99mTc-CA) and 99mTc-sulphur colloid (99mTc-SC), with respect to scintigraphy, success rate in gamma probe guided biopsy and absolute uptake in the SN. Scintigraphy was performed in six volunteers after simultaneous injection of the respective tracers in each leg. Comparison of uptake of both tracers showed a higher uptake on the 99mTc-CA side. The scintigraphic count ratio of SNs labelled with 99mTc-SC compared with 99mTc-CA was 1 to 9 28. Next, 20 patients with biopsy-proven melanoma were randomized for injection of 99mTc-CA or 99mTc-SC followed by SN biopsy. Within 20 min after the injection, focal uptake was seen in all cases of the 99mTc-CA group but in only seven of the 10 patients in the 99mTc-SC group (P < 0.05). Focal accumulations were seen in all patients of both groups after 2 h. Spill to non-SNs was seen in five of the 99mTc-CA patients and three of the 99mTc-SC patients. In all patients the SNs could be retrieved under the guidance of a gamma probe and blue dye. The uptake in the SN was significantly higher (P < 0.001) after the injection of 99mTc-CA (0.92+/-0.40%) compared with 99mTc-SC (0.34+/-0.34%). When dynamic scintigraphy is performed, 99mTc-CA is preferable. SN uptake of 99mTc-SC is less than that of 99mTc-CA but this does not adversely affect the surgical procedure.

Gamma probe guided biopsy of the sentinel node in malignant melanoma: a multicentre study.

Sentinel lymph node biopsy was attempted in 336 patients with clinically node-negative cutaneous melanoma. All patients were injected with technetium-99m labelled radiocolloid, with 108 patients simultaneously receiving vital blue dye for sentinel node identification. Sentinel lymph nodes were identified in 329 patients, giving a technical success rate of 97.9%. Metastatic disease was identified in 39 (11.9%) of the patients in whom sentinel nodes were found. Patients with negative sentinel nodes were observed and patients with positive sentinel nodes underwent comprehensive lymph node dissection. The presence of metastatic disease in the sentinel nodes and primary tumour depth by Breslow or Clark levels were joint predictors of survival based on Cox proportional hazards modelling. Disease recurrences occurred in 26 (8.8%) patients with negative sentinel lymph nodes, with isolated regional recurrences as the first site in 10 (3.4%). No patients with Clark level II primary tumours were found to have positive sentinel nodes or disease recurrences. One patient with a thin (<0.75 mm) Clark level III primary had metastatic disease in a sentinel node. Patients with metastases confined to the sentinel nodes had similar survival rates regardless of the number of nodes involved.