An effective pre-operative chemoimmunotherapy regimen against advanced gallbladder carcinoma: a case report.

We designed a chemoimmunotherapy regimen with a single dose of mitomycin C (MMC) followed by subsequent OK-432 injections, since the ability of peripheral blood mononuclear cells (PBM) to generate OK-432 activated killer cells had previously been shown to be augmented by MMC in cancer patients. We herein report on a case with far advanced gallbladder carcinoma who demonstrated a remarkable response to this chemoimmunotherapy, and thus underwent a curative resection. A 67 year-old Japanese woman was diagnosed as having gallbladder carcinoma with invasion to the liver and portal vein, as well as obvious lymph node metastasis. Since these findings suggested the tumor to be unresectable at that time, our chemoimmunotherapy regimen with MMC and OK-432 was administered. After four courses of therapy, a computed tomography (CT) scan revealed the disappearance of both tumor invasion to the surrounding organs and lymph node metastasis, which therefore prompted us to attempt a radical tumor resection. The histology of the resected specimen revealed that the majority of cancer cells had been killed by the pre-operative therapy and that only remnants of viable cancer cells were found in a part of the neck of the gallbladder and in 2 regional lymph nodes. This experience thus suggests the effectiveness of our chemoimmunotherapy regimen against gallbladder carcinoma.

Presence of elevated carcinoembryonic antigen on absorbent disks applied to nipple area of breast carcinoma patients.

BACKGROUND: Carcinoembryonic antigen (CEA) is used as a serum marker to detect and monitor the status of various kinds of malignant tumors. To determine whether CEA might be detected in secretions collected topically from around the nipple area, and whether its secretion might differ in a cancerous versus a noncancerous breast, we developed a simple method for collecting and measuring CEA, using a small cellulose membrane disk and an enzyme immunoassay. METHODS: We measured the amount of CEA excreted from the nipple area of 22 healthy control women and 32 women with unilateral breast carcinoma confirmed histologically. Secretions were collected from the nipple area by affixing a small (20 mm diameter) absorbent disk made of nitrocellulose membrane backed with filter paper to that area for 24 hours. Substances absorbed by the membrane were then subjected to an immunoassay for CEA using anti-CEA antibodies. RESULTS: In the 22 healthy subjects, a small amount of CEA (0.6 +/- 0.9 units) was secreted from each nipple, which was equally low regardless of the phase of the menstrual cycle. In contrast, 30 of the 32 women with breast carcinoma secreted significantly greater amounts of CEA from the cancerous (16.1 +/- 8.2) than the noncancerous (2.0 +/- 2.2) breast. Such a difference (14.1 +/- 8.0) in CEA excretion was not observed in the healthy controls (0 +/- 0). CONCLUSIONS: These findings suggest that such disks may provide a simple and noninvasive method of collecting trace molecules, including CEA, in skin secretions around the nipple to evaluate functional disorders of the mammary glands, particularly breast carcinoma. Additional studies are indicated in larger groups of women with various stages of breast carcinoma as well as with benign breast diseases.

Detection of cancer micrometastases in lymph nodes by reverse transcriptase-polymerase chain reaction.

There are few DNA-based studies that detect cancer micrometastases in lymph nodes. We have assayed for the specific detection of carcinoembryonic antigen (CEA)-expressing carcinoma cells in the lymph nodes of patients with gastrointestinal or breast carcinomas. A CEA-specific nested reverse transcriptase (RT)-PCR assay was optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal lymphocytes. The expression of CEA mRNA was studied in 100 carcinoma tissues, 75 normal mucosal tissues, and 15 lymph nodes from patients with cholelithiasis. Each of 117 lymph nodes from 13 patients with carcinoma was divided into two pieces: one was used for histological examination and the other for RT-PCR, and the results were compared. The sensitivity ratio was one CEA-expressing cancer cell detected in 1 x 10(5) normal lymphocytes. All carcinoma tissues and normal mucosal tissues expressed CEA mRNA, while no amplification was detected in any control lymph nodes. Thirty of 117 lymph nodes were histologically involved by carcinoma cells, and all of these yielded the expected product by RT-PCR. Of the remaining 87 histologically negative nodes, CEA mRNA was detected in 47 lymph nodes by RT-PCR. The positive rate increased from 26% by histological examination to 66% by RT-PCR. The assay by CEA-specific nested RT-PCR is not only sensitive but widely applicable for the detection of cancer micrometastases in lymph nodes. This method may lead to an earlier diagnosis and treatment of patients with subclinical lymph node metastasis.

Lymphokine-activated killer cell function of lymphocytes from regional lymph nodes in patients with gastric carcinoma.

Lymphokine-activated killer (LAK) cells generated by culture of regional lymph node cells (LNC) with interleukin 2 (IL 2) for 4 and 11 days were examined for their functional capabilities in comparison with those of peripheral blood mononuclear cells (PBM) in 25 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC for 4-day culture with IL 2 was significantly lower than that from PBM. However, the LNC-LAK cytotoxicity was markedly increased up to almost the same level as that of PBM after 11-day culture. The production of interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) from nonadherent LAK cells in LNC was also significantly reduced as compared to that from PBM 4 days after culture, when stimulated with or without tumor target, Raji cells. After 11-day culture with IL 2, however, the levels of these cytokines produced by LNC-LAK cells either with or without stimulation by tumor target were comparable to those by PBM-LAK cells, although the release of these cytokines was markedly reduced when compared to that after 4-day culture. Phenotypic analysis revealed decreased proportion of cells mediating NK activity in LNC before and 4 days after culture. CD56+ and CD57+ cells in LNC were increased after 11-day culture, although the percentages of these cells were still low as compared to those in PBM. The proportions of OKIa1+ and CD25+ cells were uniformly increased after 4 and 11-day culture in both cell populations. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBM. These results indicated the differential LAK cell function of cells from regional lymph nodes from PBM in patients with gastric carcinoma.

Lymphokine-activated killer cell function of peripheral blood mononuclear cells, spleen cells and regional lymph node cells in gastric cancer patients.

Lymphokine-activated killer (LAK) cells generated by culture of peripheral blood mononuclear cells (PBMC), spleen cells (SPC) and regional lymph node cells (LNC) with IL-2 for 4 days were examined for their functional capabilities in 29 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC was significantly lower than that from either PBMC or SPC, although there was no difference between PBMC or SPC. The induction of mRNA of interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) and the production of these cytokines in the non-adherent LAK cells from LNC were also significantly reduced compared with those from PBMC or SPC. Further, the LAK cells from LNC secreted significantly lower levels of these cytokines when stimulated with tumour target, Raji cells, although the production of these cytokines was markedly increased by stimulation with the targets in all three cell populations. Phenotypic analysis of each cell population revealed a decreased proportion of the cells mediating natural killer (NK) activity, including CD16+, CD56+, and CD57+ cells in LNC either before or after culture, although OKIa1+ and CD25+ cells were uniformly increased in all cell populations after culture. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBMC or SPC. These results indicated the differential reactivity of each lymphocyte population to IL-2 and the reduced LAK cell function of LNC compared with PBMC or SPC in patients with gastric carcinoma.

Lymphokine-activated killer cell activity of peripheral blood, spleen, regional lymph node, and tumor infiltrating lymphocytes in gastric cancer patients.

Lymphokine-activated killer (LAK) cell activity of peripheral blood mononuclear cells (PBM), spleen cells (SPC), regional lymph node cells (LNC), and tumor-infiltrating lymphocytes (TIL), induced by activation with interleukin 2 (IL 2) for 4 days, was evaluated in patients with gastric carcinoma. TIL exhibited the lowest LAK activity and the cytotoxicity of LNC was significantly lower than that of either PBM or SPC. There was no difference between PBM and SPC. Then, there were significant correlations of LAK activity among PBM, SPC, and LNC, whereas poor correlations were observed in the cytotoxicity between TIL and PBM, SPC, or LNC. Phenotypic analysis of each cell population was performed before and after activation with IL 2. Before culture, the cells mediating natural killer (NK) activity such as CD16+, CD56+, and CD57+ cells were few in LNC and TIL. However, CD56+ and CD57+ cells in TIL were increased after culture. Then, CD4+Leu8+ and CD8+CD11+ cells, which identify suppressor cell function, were not elevated in LNC or TIL, as compared to that in PBM or SPC. Further, the proportions of OKIa1+ and CD25+ cells expressing T-cell activation and IL 2 receptor were uniformly increased in all cell populations after culture. These results indicate the differential reactivity of each lymphocyte population to IL 2 and fundamental dysfunction of LNC and, especially TIL, suggesting the specific influence of the local tumor environment on the lymphocyte function in the area in patients with gastric carcinoma.

Laboratory correlates of chemoimmunotherapy with low-dose recombinant interleukin-2 and mitomycin C in patients with advanced carcinoma.

Based on our clinical findings that the ability of cancer patients to generate lymphokine-activated killer (LAK) cells was remarkably augmented after mitomycin C (MMC) administration, we designed a treatment regimen that consisted of MMC 12 mg/m2, i.v. on day 1 and recombinant interleukin-2 (IL-2) 700 U/m2, i.v. every 12 hr from day 4 through day 8. Of 29 patients with advanced carcinoma treated with this regimen, 10 had a partial response (PR) and 4 had a minor response. The correlation of hematological and immunological changes associated with this treatment with the antitumor response to this therapy was investigated. Pretreatment values of total white blood cell and lymphocyte counts, and the level of increase of eosinophil counts in responder patients who showed a PR, were significantly greater than those in nonresponder patients. However, there was no correlation between clinical response and cytotoxic activities of peripheral blood mononuclear (PBM) cells, including NK and LAK activity, and the ability to generate LAK cells after the treatment. The capacity of adherent cells in PBM to produce IL-1-beta was increased after the treatment in both responders and nonresponders, whereas IL-1-alpha production was not increased. In addition, a significant increase in the ability to produce TNF-alpha was observed only in responders, indicating the correlation of TNF-alpha production with clinical response to this therapy. Since these correlations had been reported in the previous studies using IL-2, the present results suggested that the therapeutic effectiveness of this therapy against advanced carcinoma, is due to IL-2 probably augmented by its combination with MMC. In addition, these parameters might be predictive of therapeutic efficacy of this treatment.

Neuroleptic malignant syndrome occurring after an emergency operation for traumatic duodenal perforation: report of a case.

Neuroleptic malignant syndrome (NMS) is a potentially fatal complication which may develop in psychiatric patients taking neuroleptic drugs. We report herein the successful treatment of a 33-year-old schizophrenic man, prescribed neuroleptic drugs, who underwent an emergency operation for traumatic duodenal perforation with a retroperitoneal infection. Five days after the operation, he began to demonstrate clinical features consistent with NMS such as high fever, abnormalities in vital signs, leukocytosis, and an elevated serum level of creatine phosphokinase; however, these findings were first presumed to be secondary to either the preexisting tissue injuries or to postoperative complications. A definite diagnosis of NMS was thus delayed until muscle rigidity and autonomic instability became evident. After a tentative diagnosis of NMS had been made, sodium dantrolene, a drug used specifically for the treatment of NMS, was administered and the patient's condition remarkably improved. Since NMS can be induced by either interrupting the course of neuroleptic drugs or by the additional administration of sedative drugs, and since its mortality rate is high if prompt and appropriate treatment is not carried out, surgeons should bear in mind the possibility of NMS developing postoperatively in psychiatric patients.

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C.

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical findings, we designed a treatment regimen comprised of 12 mg/m2 mitomycin C i.v. on day 1 and 700 U/m2 recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2+, CD3+, CD4+ and CD4+Leu8- cells after the first course, and CD25+ cells after either the first or second course of this treatment. The percentages of CD2+ and CD25+ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.

A trial of adjuvant chemoimmunotherapy with mitomycin C and OK-432 for stage III gastric carcinoma.

We previously found that the ability to generate cytotoxic cells induced by in vitro activation of peripheral blood mononuclear cells (PBM) with OK-432, a bacterial immunopotentiator, was markedly increased following intravenous administration of a single dose of mitomycin C (MMC) in cancer patients. On the basis of this clinical finding, we designed a treatment regimen that consisted of MMC 12 mg/m2 intravenously on day 1 and OK-432 5 Klinische Einheit (KE) intradermally on days 6, 8, and 11, when the generation of OK-432 activated killer cells had been shown to be significantly augmented. Then, it was followed by long-term tegafur. Fifteen patients with stage III gastric carcinoma who had undergone curative resection were treated with the above regimen. The survival of these patients was significantly better than that of 26 comparable stage III patients concurrently treated with MMC 12 mg/m2 alone, followed by long-term tegafur (P less than 0.01). The results indicate that OK-432 combined with MMC may be effective against stage III gastric carcinoma, when these agents are used probably in an appropriate combination.

Enhanced induction of lymphokine-activated killer activity after lentinan administration in patients with gastric carcinoma.

In 15 patients with gastric carcinoma, peripheral blood mononuclear cells (PBM) were obtained serially before and 3, 5 and 7 days after lentinan administration. The generation of lymphokine-activated killer (LAK) activity, induced by in vitro activation of PBM with interleukin 2 (IL 2), was significantly augmented 5 days after a single intravenous dose of 2 mg lentinan, when compared with that before lentinan injection. Natural killer (NK) activity of PBM was also significantly enhanced 7 days after the drug injection. However, the distribution of lymphocyte subsets exhibited no significant change following lentinan administration.

Correlation of eosinophilia with clinical response in patients with advanced carcinoma treated with low-dose recombinant interleukin-2 and mitomycin C.

On the basis of our clinical findings that the ability of cancer patients to generate lymphokine-activated killer cells became markedly augmented after mitomycin C administration, we designed a treatment regimen comprising mitomycin C 12 mg/m2, i.v. on day 1 and recombinant interleukin-2 700 U/m2 (8000 IU/kg), i.v. every 12 h from day 4 through day 8. The treatment course was repeated at almost 7-day intervals. Altogether 33 patients with advanced carcinoma, including mainly gastrointestinal carcinoma, were treated with this regimen. Of these, 10 had a partial response (PR) and 4 had a minor response (MR). Since eosinophil counts peaked 1 day after either the first or second course of the therapy, the posttreatment values were compared to each pretreatment level, with regard to the clinical antitumor response to this treatment. When patients who showed PR were defined as responders, absolute eosinophil counts and the percentage of eosinophils in responders after both the first and second courses of the therapy were significantly greater than each pretreatment value or the posttreatment level in nonresponders. Further, these findings were almost identical, when both PR and MR were considered to be a true remission and therefore patients who exhibited PR or MR were defined as responders, although the difference between posttreatment levels of eosinophils in responders and nonresponders was not significant at the second course. These results indicate that eosinophilia induced by this treatment correlates with the clinical response to this therapy.

Enhanced production of interleukin 1 and tumor necrosis factor by peripheral monocytes after lentinan administration in patients with gastric carcinoma.

The effect of intravenous administration of lentinan, an immunopotentiating polysaccharide, on the production of interleukin 1-alpha (IL 1-alpha), interleukin 1-beta (IL 1-beta) and tumor necrosis factor-alpha (TNF-alpha) by monocytes in peripheral blood mononuclear cells (PBM) was studied in patients with gastric carcinoma. Peripheral blood samples were obtained from 10 patients before and 3, 5 and 7 days after a single dose of 2 mg lentinan injection. The ability of monocytes in PBM to produce IL 1-alpha was significantly augmented 3 and 5 days after lentinan administration, as compared with that before treatment. IL 1-beta production was also significantly increased 3, 5 and 7 days after the drug injection. Further, the capacity to produce TNF-alpha was significantly enhanced 3, 5 and 7 days after the drug administration. Thus, it is likely that the augmentation of these cytokine's production may contribute to the antitumor action of lentinan in patients with gastric carcinoma.

Changes on levels of B6 vitamin and aminotransferase in the liver of diabetic animals.

We measured aminotransferase activity and vitamin B6 content in the livers of diabetic mice. Two different types of mice were used for the measurements, spontaneously non-obese diabetic (NOD) or alloxan-induced diabetic (Allo) mice, and control mice were either non-diabetic NOD or Institute of Cancer Research (ICR). The liver of diabetic mice had more aspartate aminotransferase (AST) activity than those of normal mice. The diabetic livers also had more vitamin B6 than did normal livers, and pyridoxamine (PM) levels were particularly high but pyridoxal (PL) levels were not. ICR livers showed hepatic alanine aminotransferase activities inversely correlated with blood glucose concentrations, while diabetic livers did not. The abundance of AST and B6 in the diabetic liver is consistent with the great need for gluconeogenic substrate there. This is understandable in that most aminotransferases require B6 vitamins, and especially the correlation between s-AST and PM levels was recognized in the diabetic liver. Conversely, the AST and PM levels were negatively correlated in normal mice. A metabolic shift towards gluconeogenesis apparently produces more B6 and PM while it induced holo-AST synthesis.

The effect of recombinant interleukin 2 in combination with mitomycin C on advanced cancer.

We recently discovered that the ability of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C (MMC) administration. Based on our clinical findings, we designed a treatment regimen comprised of MMC 12 mg/m2 given intravenously on day 1 and recombinant interleukin 2 (rIL 2) 700 U/m2 given intravenously every 12 hr from day 4 through day 8, when the generation of LAK cells had been shown to be markedly increased. Ten patients with various advanced carcinomas for which standard therapy had failed or no standard therapy was available, were treated with this regimen. Of these ten, three had a partial response and three others had a minor response. Fevers were common and anemia occurred in four patients, but nevertheless, severe toxicity was not encountered. These results indicated that rIL 2 in combination with MMC might be effective against advanced carcinoma without causing severe toxicity when these drugs are used in an appropriate combination.

Augmentation of the generation of lymphokine-activated killer cells after a single dose of mitomycin C in cancer patients.

The effect of mitomycin C administration on the generation of cytotoxic cells, induced by in vitro activation of peripheral blood mononuclear cells (PBM) with interleukin-2, was studied in patients with various carcinomas. The ability of PBM to generate lymphokine-activated killer (LAK) cell activity against Raji cell targets was significantly augmented 5 and 7 days after a single intravenous dose of 12 mg/m2 mitomycin C, when compared to that of PBM obtained before mitomycin C injection. Further, LAK cell activity against autologous tumor cells was also significantly increased after the drug administration. The distribution of lymphocyte subsets exhibited a significant increase in the percentage of CD3+ cells after injection, with the elevation of the CD4/CD8 ratio. Furthermore, the proportion of the CD4+ Leu8+ subpopulation, which identifies inducers of suppression, was significantly reduced. Thus, the decrease in the proportion of suppressor-inducer subsets of PBM might be at least partially, responsible for the augmented generation of LAK cells after mitomycin C administration.

Aminotransferase activity in the liver of diabetic mice.

Enzyme activity in the livers of mice was studied in examining the metabolic disturbances of diabetes. Spontaneously non-obese diabetic (NOD) mice, mice with alloxan-induced diabetes (Allo), and control ICR mice were used. As NOD mice undergo a spontaneous pathogenic process over time, younger and older NOD mice were compared (non-diabetic and diabetic) as were control ICR mice. Two liver enzymes became more active with age, aspartate aminotransferase (AST) and alanine aminotransferase (ALT). AST activity increased more in the hyperglycemic mice, i.e., the diabetic NOD and the Allo mice, than in the normoglycemic group, i.e., the ICR and non-diabetic NOD mice. Abnormally high AST activity was seen in the cytosolic fraction of the liver but not in the mitochondrial fraction. The changes in enzyme activity in diabetic mice were independent of any age-associated changes. The higher AST levels in diabetic mice are thought to be consistent with their greater need for gluconeogenic substrate. AST showed a more notably higher increase than did ALT in this study, and lactate dehydrogenase showed no significant changes.