Ex vivo activated memory T-lymphocytes as adoptive cellular therapy of human soft-tissue sarcoma targets with potentiation by cis-diamminedichloroplatinum(II).

Autolymphocyte therapy (ALT) is tumor-specific, adoptive cellular therapy of neoplastic disease using nonspecific ex vivo activation of autologous peripheral blood lymphocytes (PBL), which are composed primarily of memory T-cells (ALT-cells) and are active in patients with metastatic renal cell carcinoma and melanoma. Ex vivo pretreatment of tumor target cells with certain chemotherapeutic agents can enhance susceptibility to lysis by antitumor lymphocytes. To determine if cis-diamminedichloroplatinum(II) (CDDP) enhances ex vivo antitumor cytotoxicity of ALT-cells and if this lysis is mediated by T- and/or NK-cells and is human leukocyte antigen (HLA)-restricted, human soft tissue sarcoma (STS) target cells were derived from primary and metastatic surgical specimens and were incubated with and without CDDP. ALT-cells were prepared from autologous PBL obtained prior to surgery. Primary (PSTS) and metastatic (MSTS) target cells from each group were labelled with chromium 51 (51Cr) and used as targets for ALT-cells, CD45-depleted ALT-cells, CD56 (NK)-depleted ALT-cells, and PBL in a standard (4-hour) and delayed (18-hour) 51Cr release assay. Interferon-gamma (IFN-gamma) release was measured as an indication of antitumor effect and recognition by the noncytolytic lymphocytes in ALT-cells. Primary tumor target cells incubated in CDDP showed enhanced lysis as measured by the 51Cr release assay compared to non-CDDP-treated controls. Metastatic tumor target cells showed less lysis than the primary targets, although this was enhanced by pretreating metastatic tumor targets with CDDP. Lysis of all tumor targets was significantly greater when ALT-cells were used as the effector cells rather than PBL. Depletion of memory T-cells abrogated ex vivo lysis. Depletion of NK cells (CD56+) affected ex vivo lysis of autologous targets during the 4-hour but not the 18-hour assay. Ex vivo ALT-cell lysis and IFN-gamma release against only the autologous tumor targets confirmed tumor-specificity in one patient. Restriction of ALT-cell lysis and IFN-gamma release against HLA-A2+ autologous and one allogeneic HLA-A2+ STS tumor target, but not other non-STS targets, was demonstrated in another patient. These data suggest that CDDP may help render STS susceptible to tumor-specific, immune-mediated attack and that the combination of ALT and CDDP may lead to effective tumor-specific chemoimmunotherapy in patients with metastatic STS.

The role of microvascular surgery in limb-sparing procedures for malignant tumors of the knee.

Limb-sparing surgery has proven to be a feasible alternative to amputation for treatment of malignant tumors about the knee. Microvascular surgery and the possibility of providing healthy, stable soft-tissue coverage to the affected limb have expanded the possible role of limb-sparing techniques. Microvascular free flaps were utilized in 26 patients undergoing resection for large malignant tumors of the knee or adjacent to the knee. Patients were then followed for a minimum of 3 years to a maximum of 7 years and evaluated for tumor recurrence and limb function. Survival and disease-free interval were 68 and 77 percent, respectively, in stage II patients and only 50 and 0 percent, respectively, in stage III patients. Function, as determined by the MSTS functional evaluation system, was found to be fair to good in all patients. Microvascular free-tissue transfer has proven to be a valuable adjunct in limb-sparing surgery.

Primary tumors of the thoracic skeleton.

From 1975 to 1990, eighty-nine primary tumors of the thoracic skeleton; ribs, sternum, scapula, clavicle, and thoracic spine, were treated. Forty-four tumors (49%) were benign lesions. Forty-five tumors were malignant and were proportionately distributed amongst the five sites. The most common malignancies were Ewing's sarcomas, chondrosarcomas, plasmacytomas, osteogenic sarcomas, and lymphomas. All patients with Ewing's sarcomas were treated with combination chemotherapy, surgical resection, and radiation therapy for those with residual disease after surgery. Only one patient has died of disease. Patients with primary chondrosarcomas were treated by surgery alone and all are free of disease or have died without disease. Patients with solitary plasmacytomas or primary lymphomas of bone were treated with radiation therapy initially. Half the patients developed systemic disease. The patients with osteogenic sarcomas included several with radiation induced lesions and Paget's osteosarcoma and all but one died of disease.

Role of angiography in limb-tumor surgery.

Routine preoperative angiograms obtained in 97 consecutive patients with bone and soft-tissue tumors of the extremities were retrospectively reviewed to determine the role of angiography in surgical planning. Some degree of major vessel displacement was demonstrated in 51 patients. Encasement of major vessels, which precluded limb-salvage surgery, was seen in five of 76 bone tumors and in three of 21 soft-tissue tumors. All five patients with osteochondromas had associated vessel occlusion or compromise. For surgical planning, the demonstration of adequate anastomoses in the arches of the hands and feet is important, as branches of brachial and popliteal arteries may have to be sacrificed during limb-tumor surgery. Angiographic findings indicated or supported the need for a vascular surgeon in five cases in this series. Preoperative angiography provides important information with regard to the status of the vasculature and therefore is essential in the management of bone and soft-tissue tumors of the extremities.

The acute effects of hematoporphyrin derivative photoradiation on tumor and liver capillary blood flow.

The acute effects of photoradiation after administering hematoporphyrin derivative (Hpd) on capillary blood flow were studied in intrahepatic tumors and normal liver. The tumors were solitary Walker carcinosarcomas implanted within the livers of Sprague-Dawley rats. Capillary flow was measured by a laser doppler monitor with its probe positioned over the tumor or over normal liver. Within a minute after intraportal Hpd injection (1.7 mg), capillary flow in the tumors began to decrease. Minimal levels of flow were maintained for as long as 15 minutes after Hpd injection with no observed recovery of flow back to control levels. Ratio of minimal flow/control flow averaged 0.36. Similar results were seen in studies on normal liver tissue. These studies demonstrate the extremely rapid vasoactive effects caused by photoradiation of Hpd. Vasoconstriction, vascular stasis and ischemia have proven to be important mechanisms in producing tumor cell destruction by photodynamic therapy.

Augmentation of the effect of doxorubicin with low-dose tumor necrosis factor in experimental liver metastasis.

The antitumor activity of recombinant human tumor necrosis factor was studied in vivo as a single agent and in combination with a conventional chemotherapeutic agent. Dosages of tumor necrosis factor of 100 micrograms, 50 micrograms, and 25 micrograms were injected intraportally in Sprague-Dawley rats containing hepatic implants of Walker carcinosarcoma. An effect on the tumor was seen but was associated with a significant acute mortality. Lower dosages of tumor necrosis factor, 10 micrograms, 5 micrograms, and 1 microgram, administered with 10 mg/kg of doxorubicin (Adriamycin) significantly enhanced the antitumor effect of doxorubicin without an acute mortality. This suggests that lower dosages of tumor necrosis factor with conventional chemotherapy may augment the latter's effect without any added toxicity.

Evidence that epinephrine acutely redistributes blood flow to experimental intrahepatic tumors.

Tumor microcirculation was studied in Sprague-Dawley rats with solitary intrahepatic implants of Walker carcinosarcoma tumors. Thioflavine S (TS), a fluorescent dye that stains capillary endothelium acutely, was injected intraportally or intra-arterially in order to demonstrate patterns of blood flow through normal liver tissue and through tumor. The dye was given immediately after intraportal injection of 10 micrograms of epinephrine in some animals and 3 minutes after the epinephrine in others. Control animals received TS alone. Additional animals were given TS immediately after intra-arterial epinephrine. The degree of resulting fluorescence in tumor and liver was graded subjectively from 0 to 3+. In the controls and in animals receiving TS 3 minutes after epinephrine, fluorescence in the centers of tumors was absent or, at most, faintly present. In contrast, all animals given epinephrine either intraportally or intra-arterially immediately before the dye showed intense fluorescent staining within the centers of the tumors. Subjective grading averaged 0.6 +/- 0.1 in the controls, 2.2 +/- 0.1 in those receiving intraportal epinephrine immediately before TS, and 1.0 +/- 0.3 in those receiving TS 3 minutes after epinephrine. Results were significantly higher in the latter group (p less than 0.01). Subjective grading in animals receiving intra-arterial epinephrine immediately before TS averaged 2.5 +/- 0.3. These experiments confirm previous studies in this laboratory that demonstrated an acute short-lived redistribution of blood flow into the centers of intrahepatic tumors after administration of epinephrine.

Increased capillary flow in intrahepatic tumors due to alpha-adrenergic effects of catecholamines.

Chemotherapy of liver metastases is dependent on adequacy of tumor microcirculation. Attempts have been made, experimentally, to improve tumor blood flow with appropriate vasoactive agents. Capillary blood flow within intrahepatic Walker carcinosarcomas and normal liver were measured with a laser doppler needle probe. Tumor capillary blood flow increased briefly but significantly with intraportally administered epinephrine. This effect was blocked by phenoxybenzamine but not by propranolol. The response of capillary flow in normal liver to epinephrine was dose related, with decreased flow with higher doses, and slight increase with lower doses. Norepinephrine and phenylephrine produced brief increases in capillary flow in tumor and liver, and isoproterenol caused a decreased flow. In studies with injected silicone rubber (Microfil, Canton Bio-Medical Products, Boulder, CO) performed previously, and on electron microscopic examination, there was evidence of increased vascular filling within central areas of tumors after epinephrine. These studies suggest that the brief but potent effect of epinephrine on tumor capillary flow could be useful in improving delivery of chemotherapeutic agents to liver tumors.

Enhancement of tumor blood flow and tumoricidal effect of doxorubicin by intraportal epinephrine in experimental liver metastasis.

Laser Doppler capillary perfusion monitoring was performed in 25 Sprague-Dawley rats with solitary intrahepatic implants of Walker carcinosarcomas. Doses of intraportal epinephrine from 1 to 10 micrograms produced an immediate rise in capillary blood flow, reaching peak levels by 16 s. The increased flow lasted an average of 57 s. Because epinephrine selectively increased tumor blood flow, studies were performed to determine if enhancement of the chemotherapeutic effect of doxorubicin could be achieved. Forty-two rats with seven-day hepatic implants were treated sequentially with intraportal epinephrine (10 micrograms) and doxorubicin hydrochloride (10 mg/kg) or doxorubicin alone. After only two courses of chemotherapy, a significant difference in necrosis and cell viability was obtained in those animals treated with epinephrine intraportally prior to doxorubicin infusion. Enhancement of the tumoricidal effect of chemotherapy can be achieved by manipulating the blood flow to developing tumors pharmacologically.

Electrocautery: effects on steroid receptors in human breast cancer.

The determination of steroid receptors in human breast cancers has assumed increasing importance over the past several decades. Improper handling of the specimens could affect results obtained. This study details the effects excessive levels of heat that occur with the use of electrocautery can have on steroid receptor quantities and localization. Twelve resected primary and metastatic human breast cancers were analyzed for cytoplasmic and nuclear receptors by biochemical analysis. In addition, steroid binding was determined by direct fluorescent histochemical techniques. To a portion of each resected specimen a Boviec was applied to simulate electrocautery resection. Analysis of the different portions of the same tumor revealed that there was a decrease in measurable cytoplasmic receptor in all cauterized specimens and a concomitant increase in the nuclear receptor. A similar shift in steroid binding was noted in all the specimens analyzed by fluorescent histochemical techniques. The results of this study show that the application of excessive heat to human breast cancers will lead to false negative biochemical steroid receptor determination by shifting the receptors intranuclear.

Tamoxifen treatment failures in hormonally responsive breast cancers. Correlation with steroid receptors.

Clinical correlation of treatment response with estrogen (ERP) and progesterone (PgRP) receptors in 81 patients revealed a remission rate of 76% if both receptors were present in the tumor. The response rate was 89% in those patients with positive receptors in whom an endocrine ablative procedure was performed and was 63% in those patients treated with tamoxifen, suggesting that the latter modality may not be effective in the treatment of some hormonally responsive tumors. An assay to determine receptor sensitivity to tamoxifen was developed and correlated with clinical response to tamoxifen treatment. Of the 56 human breast tumors analyzed, 29 tumors were ERP- and PgRP-positive and ten of these were tamoxifen-insensitive. Sixteen tumors were ERP- and PgRP-negative and tamoxifen-insensitive. Of the remaining 11 tumors, four were tamoxifen-sensitive. Nineteen of the 56 patients whose tumors were analyzed were treated for advance breast cancers with tamoxifen. Twelve patients who were ERP- and PgRP-positive and tamoxifen-sensitive responded to this modality of treatment. Seven treatment failures were noted in this group all of whom were ERP-positive and tamoxifen-insensitive. Endocrine ablation failed either prior or subsequently in three of these patients all of whom were PgRP-negative. Two patients who were ERP- and PgRP-positive and tamoxifen-insensitive subsequently responded to an alternative antihormonal treatment.

Benign ulceration of the cecum presenting with massive hemorrhage: report of a case and review of the literature.

Massive gastrointestinal hemorrhage secondary to benign cecal ulceration is a rare occurrence. The diagnosis of bleeding from this benign condition is rarely entertained and is often a finding on a pathologic examination. A case of massive hemorrhage from a cecal ulcer is presented, and the literature is reviewed. The role of angiographic demonstration of the lesion, if angiography is readily available, is emphasized, but surgery is the definitive therapy.

Immunohistologic and histochemical methods for detection of steroid binding in breast cancer: a reappraisal.

A review of the current literature on immunohistologic and histochemical methods for the detection of steroid hormone binding sites in breast cancer, reveals that many, but not all of the criteria for establishing hormone-receptor binding interactions have been met. These include tissue specificity, binding between labeled ligands and soluble receptor in vitro, correlations between histochemical and biochemical assays, as well as between histologic procedures and tumor hormone responsiveness. However, histochemical binding phenomena do not appear to follow classical receptor dogma in regard to the concentration of ligand required, or specificity of binding as determined by competitive binding assays. It is concluded that these histologic techniques may be detecting classical receptor that may be reacting differently than would be expected from biochemical analyses, Types II and III binding sites, and/or organelle and membrane-bound receptors. Certainly no current method should presently be promoted as a laboratory method for the detection of classical receptor. New immunocytologic procedures employing specific, antireceptor sera currently under development, may obviate many of the criticisms leveled against earlier methods.

Histochemical assay of estrogen and progesterone receptors in breast cancer: correlation with biochemical assays and patients' response to endocrine therapies.

Estrogen (ER) and progesterone (PgR) receptors were assayed by histochemistry in primary, recurrent, and metastatic breast cancer. Ligand-conjugates composed of 17 beta-estradiol and 11 alpha-hydroxyprogesterone covalently linked to bovine serum albumin and labelled with fluorescein isothiocyanate were employed. Results were compared with those of conventional biochemical receptor assays and correlated for ER in 92% of 314 tumors and for PgR in 86% of 86 specimens. ER and PgR determinations by both assay systems were correlated with clinical response to various endocrine therapies in 40 women with Stage IV disease. The histochemical assay enabled successful prediction of response in 80% of cases including eight which could not be fully analyzed biochemically.

Carcinoma of the cheek mucosa. A retrospective analysis.

A 16 year experience with 121 patients treated for squamous cell carcinoma arising in the cheek mucosa is described. Treatment was almost exclusively surgical, and 5 year determinate cure rates ranged from 77 and 65 percent in stage I and II lesions to 27 and 18 percent in those with stage III and IV lesions, respectively. Twenty-four percent of previously treated patients remained alive and well at 5 years. The 42 percent overall salvage rate was low, reflecting the high proportion of patients who developed cervical metastasis during the course of the disease. In an attempt to improve local control, we now advocate irradiation of the primary tumor and neck after aggressive resection with radical neck dissection in any patient who has clinical evidence of nodal involvement or other unfavorable findings.

Histochemical analyses of steroid hormone receptors in breast and prostatic carcinoma.

Histochemical analyses estrogen (ER) and progesterone (PgR) receptors in breast cancer were statistically correlated with results of dextran-coated charcoal (DDC) and sucrose gradient assays. Correlated for ER was 91% of 363 cases, and for PgR 88% of 255 specimens. Breast cancer ER/PgR positivity by histochemistry correlated with a favorable clinical response to endocrine therapies in 72% of 25 cases, while ER/PgR negativity correlated with a lack of response in 96% of 22 cases with Stage IV disease. Nuclear ER/PgR correlated with a poor response to therapy in 8 of 12 patients. An in vitro technique to detect nuclear translocation of ER revealed two groups of ER positive cases, with 11 of 17 exhibiting translocation and 6 not displaying translocation. In prostatic carcinoma, 72% of 65 men were positive for ER and/or androgen receptor. Comparison of specimens obtained without and with electrocautery revealed a preponderance of nuclear binding in the latter, suggesting heat-induced nuclear translocation of receptor. coumestrol, a naturally fluorescent, entirely unaltered estrogen was also used for histochemical detection of ER. Results correlated with ER by DCC in 87% of 61 breast cancers. Coumestrol was additionally used to visually observe receptor and nuclear translocation of ER in intact whole cells in culture.

The role of progesterone receptors in the management of advanced breast cancer.

The identification of steroid receptors in human breast cancer tissues has provided biochemical markers to predict hormone responsiveness. Analysis of tumors from 328 patients for the estrogen receptor protein (ERP) revealed significant levels in 225 patients (68%). Fifty-five patients with advanced disease were subjected to hormonal manipulation. Thirty patients underwent ablative surgery consisting of adrenalectomy or adrenalectomy and oophorectomy. Twenty-five patients were treated with additive therapy utilizing the new antiestrogen Tamoxifen. The ECOG criteria for an objective response were applied to all patients. Fifteen of the 24 estrogen receptor-positive patients (63%) subjected to endocrine ablation responded. Eleven of twenty (55%) ERP-positive patients responded to Tamoxifen therapy. Only one ERP-negative patient responded in the total group. Simultaneous analysis of these same specimens for the progesterone receptor protein (PgRP) revealed that 151 patients were ERP and PgRP positive (46%); 74 (23%) were ERP positive and PgRP negative; 90 (27%) were ERP and PgRP negative, and 13 (4%) were ERP negative and PgRP positive. In the two treatment groups the overall response rate when both receptors were positive was 77% with a higher percentage of responders in the endocrine ablative group (88% vs. 64%). In addition, a greater duration of response was achieved in those patients treated with endocrine ablation.

Group work in a hospital waiting room.

For students who have determination, field-work placements offer an opportunity not only to learn, but to develop innovative services. The authors give a personal account of how, as students, they initiated a program of group work in a hospital waiting room.