Feasibility of delta-shaped anastomoses in totally laparoscopic distal gastrectomy.

BACKGROUND: Delta-shaped (DS) anastomosis is a new reconstruction method for totally laparoscopic distal gastrectomy (TLDG) using a linear stapler. We evaluated the feasibility of using this method for TLDG. METHODS: A retrospective analysis was performed in 114 patients who underwent TLDG with DS anastomosis. Twenty-four patients reconstructed with a Roux-en-Y (RY) anastomosis during the same period were analyzed as control subjects. RESULTS: The patient characteristics of DS and RY anastomoses were slightly different in terms of tumor location and extent of lymph node dissection, since this was not a prospective comparative study. Blood loss, postoperative complication rate and postoperative hospital stay were not different between the two groups. There was only 1 case of anastomotic leakage, and no case of anastomotic stricture after DS anastomosis. The length of the operation using DS anastomosis was significantly shorter than for RY anastomosis. The rates of body weight loss were not significantly different at 1 year after the operation. CONCLUSIONS: Although this was a small retrospective analysis, DS anastomosis was feasible, required a shorter operation time, and had no associated complications. This method can therefore be recommended as a standard procedure for TLDG.

Effect of heat treatment on tumor cells and antitumor effector cells.

We examined the effects of heat treatment on tumor cells and antitumor effector cells in order to investigate the combined effects of hyperthermia and immunotherapy including adoptive immunotherapy. Plasmacytoma MOPC104E syngeneic to BALB/c mice was used as the tumor cell line, while fresh spleen cells immunized to MOPC104E (IM-FSC) and interleukin-2-cultured lymphocytes induced in vitro from IM-FSC (CL) were used as the effector cells. Tumor cells or effector cells were heat-treated in a water bath at 42 degrees C for 30 or 60 min. Tumor cells heat-treated at 42 degrees C for 30 min grew temporarily and then regressed in the tumor transfer test, whereas untreated tumor cells showed no regression under any conditions. Furthermore, fresh spleen cells of mice inoculated with heat-treated tumor cells from regressed tumors showed marked tumor-neutralizing activity. The antitumor effects of CL were markedly inhibited by heat treatment according to the results of the tumor-neutralizing test and the 51Cr release assay, whereas heat treatment had little influence on the antitumor activity of IM-FSC. However, the neutralizing activity of effectors and the killing activity of CL against heat-treated tumor cells were both markedly augmented, since the susceptibility of the tumor cells to the antitumor effector cells was augmented by heat treatment. These results suggest that heat treatment of tumor cells augments the antitumor effects of IM-FSC and CL, hence we speculate that hyperthermia augments the effects of immunotherapy including adoptive immunotherapy.

Ratio of motor nerve conduction velocity to F-wave conduction velocity in diabetic neuropathy.

OBJECTIVE: To investigate the usefulness of a new parameter, the ratio of motor nerve conduction velocity to F-wave conduction velocity (M/F ratio), for the differential diagnosis of diabetic neuropathy. RESEARCH DESIGN AND METHODS: Nerve conduction studies were conducted in 95 patients with diabetic neuropathy, 44 nondiabetic patients with peripheral neuropathy, and 24 normal control subjects. Nondiabetic patients with neuropathy were grouped by clinical diagnosis as follows: segmental demyelination (n = 15), axonal neuropathy (n = 11), alcoholic polyneuropathy (n = 4), and other polyneuropathy (n = 14). Motor nerve conduction velocity (MCV) of post-tibial nerves, sensory nerve conduction velocity (SCV) of sural nerves, and F-wave conduction velocity (FWCV) of post-tibial nerves were measured by standardized techniques. The M/F ratio was calculated from these measurements. RESULTS: The MCV and SCV of diabetic patients were significantly slower and the M/F ratio was significantly lower than those of normal subjects: MCV, 43.7 +/- 5.4 vs. 47.1 +/- 2.9 m/s, P < 0.001; SCV, 44.7 +/- 11.1 vs. 48.3 +/- 5.7 m/s, P < 0.05; M/F ratio, 0.84 +/- 0.09 vs. 0.90 +/- 0.06, P < 0.001. The FWCV of nondiabetic patients with neuropathy was significantly slower (40.0 +/- 6.3 vs. 48.3 +/- 4.0 m/s, P < 0.001) and the M/F ratio was significantly higher (1.04 +/- 0.12, P < 0.001) than that of normal subjects, respectively. Although MCV, SCV, and FWCV were correlated with age in normal control subjects, the M/F ratio was independent of age in the diabetic as well as the nondiabetic patients with neuropathy. CONCLUSIONS: Results suggest that the M/F ratio, which is influenced by the neuronal damages in the distal segment of peripheral nerves, is useful in the differential diagnosis of diabetic neuropathy.

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from breast cancer.

We studied the therapeutic effect of OK-432 combined with adoptive immunotherapy in 19 cases of liver metastases from breast cancer. Of the 14 patients who received intraarterial OK-432 injection and transfer of cultured lymphocytes, 9 responded to this therapy, whereas no patients responded to intravenous administration. The minimum cell number for a therapeutic response was 8 x 10(8) cells. Metastatic lesions other than those in the liver regressed after therapy in 4 patients. The serum carcinoembryonic antigen level paralleled the therapeutic effect. There were no severe side-effects accompanying this therapy. These results indicate that intraarterial adoptive immunotherapy combined with OK-432 is effective as a new therapeutic approach against liver metastases from breast cancer.

Factors influencing the response and survival of patients with liver metastases from breast cancer receiving OK-432-combined adoptive immunotherapy.

The response and survival of 26 patients with liver metastases from breast cancer, who received OK-432-combined adoptive immunotherapy from 1984 to 1990, were evaluated. OK-432-combined adoptive immunotherapy was comprised sequential treatment via the hepatic artery with a streptococcal preparation, OK-432 (1-5 KE), and adoptive transfer of lymphocytes expanded in T-cell growth factor and sonicated tumor extract antigen. Seventeen (65%) patients responded to the therapy. The median survival time of all patients after treatment was 13 months (range, 2-63 months). Of the 20 prognostic factors analyzed, performance status (PS) alone was related to response (P less than 0.01). The response rate of the patients with a PS of 0-2 was 83% but only 25% in those with a PS of 3 or 4. In univariate analysis, 11 factors significantly influenced the survival: tumor response; size of primary tumor; menopausal status; PS; serum bilirubin, albumin, lactate dehydrogenase and glutamate-oxalate transaminase (aspartate aminotransferase); the extent of liver involvement; and the number and the proliferation rate of transferred lymphocytes. The MST was 22.8 months for the responders versus 2.8 months for the nonresponders (P less than 0.01). In multivariate analysis, the most important factor associated with survival was the tumor response, as well as PS, liver involvement, lactate dehydrogenase and albumin. These results suggest that OK-432-combined adoptive immunotherapy can be considered a candidate for a randomised control study and these factors should be used for stratification.

Binding and internalization of human immunoglobulin G conjugated with melphalan (K18) to human tumor cell lines.

The binding and internalization of melphalan-conjugated human immunoglobulin G (K18) to human tumor cell lines were studied using 14C-K18 (IgG conjugated with 14C-melphalan) and were compared with those of 14C-melphalan. K18 and melphalan dose-dependently bound to tumor cells, and the saturation binding analysis revealed a receptor-mediated binding of K18 but not of melphalan, to tumor cells. Intracellular distribution of 14C-K18 differed from that of 14C-melphalan, but a DNA unwinding experiment using a hydroxylapatite column showed that 14C-melphalan or 14C-K18 bound to DNA. These results suggest that after being bound to receptors K18 may be internalized in a macromolecular form and degraded into peptide binding melphalan. Moreover, the quantity of K18 that bound to 7 different human tumor cell lines was significantly larger than those that bound to lymphocytes of normal donors. These results suggest that K18 may be beneficial for cancer chemotherapy.

The inhibitory effect of a conjugate of human immunoglobulin G and melphalan, K18, on DNA synthesis of human tumor cells.

K18 is a conjugate of human immunoglobulin G and melphalan and has been reported to accumulate selectively in the tumor. In the present study, the in vitro antitumor effect of K18 was assessed in a total of 107 fresh human tumors by DNA synthesis (3H-thymidine incorporation) inhibition assay, and compared with that of other drugs. Human tumor cells showed a variety of sensitivities to K18: gastric, breast, pancreatic, liver or ovarian cancer cells were comparatively sensitive to K18, while esophageal or colorectal cancers were insensitive. These results suggest that the conjugation of melphalan with IgG did not reduce the antitumor activity of melphalaln and that K18 may be applied in cancer chemotherapy instead of melphalan.

In vivo inhibitory effect of a conjugate of immunoglobulin G and melphalan, K18, on human tumors transplanted in nude mice.

Various human tumor lines, including gastric, colonic, esophageal, pancreatic, lung and breast cancer, were transplanted in nude mice, and the effect of K18 (IgG conjugated melphalan) on them was assessed and compared with that of melphalan. Melphalan (1 mg/kg) or K18 (100 mg/kg) were administered to mice for 28 days. The tumor growth was significantly inhibited in 5 out of 6 tumors by both agents, although only the esophageal line was insensitive to both agents. Significant loss of body weight was observed in mice after treatment. K18 had no influence on body weight. These results suggested that K18 may be useful in cancer chemotherapy.

Anticancer chemosensitivity profiles of human breast cancer cells assessed by in vitro DNA synthesis inhibition assay.

The present study was designed to assess the profile of the chemosensitivity of breast cancer cells and to screen effective agents for combination regimens. Chemosensitivity to anticancer agents was assessed by the 3H-thymidine incorporation assay, as the rate of inhibition of DNA synthesis in 145 samples (88 primary and 57 metastatic or recurrent lesions) from 136 patients with breast cancer. The correlations of the anticancer agents with various clinicopathological factors were analysed. The effectiveness of the agents was classified as a rate of inhibition on log scale as follows: highly sensitive (> or = 30%), moderately sensitive (25-30%), slightly sensitive (20-25%), resistant (< 20%). The chemosensitivity of breast cancer showed variations according to tumor location: primary lesions seemed to be slightly sensitive to carboquone (CQ), adriamycin (ADR), and cytosine arabinoside (Ara-C); nodal involvement was moderately sensitive to CQ and slightly sensitive to Ara-C, 5-FU, ADR, mitomycin-C (MMC), and cisplatin (CDDP); malignant effusions were highly sensitive to ADR, moderately sensitive to CQ, and slightly sensitive to Ara-C and CDDP; local recurrences were slightly sensitive to Ara-C, CQ and 5-FU; vincristine (VCR) and nimustine chloride (ACNU), however, seemed to be ineffective against breast cancer. There were significant correlations in chemosensitivity between most agents, but no correlation was found between 5-FU and CDDP, 5-FU and ACNU, MMC and VCR, ADR and CDDP, ADR and VCR, and ADR and ACNU. There were no differences in chemosensitivity between stages of primary lesions or between estrogen receptor-positive and -negative tumors. In 10 patients, simultaneous nodal involvement was more sensitive to the agents than were primary lesions, and the correlation of chemosensitivity to ADR and CQ between such lesions was significant. On the other hand, there was no significant difference or correlation of chemosensitivity between the original lesions and recurrent ones after chemotherapy. The heterogeneity and homogeneity in the chemosensitivity of breast cancer suggested not only the necessity of patient-specific chemotherapy based on a sensitivity assay, but also the usefulness of choosing agents for widely-applicable combination regimens against breast cancer.

Real-time sonography of palpable breast masses.

In order to evaluate the clinical usefulness of an electronic real-time linear array scanner in breast diseases, ultrasonography was performed in 148 cases of histologically confirmed palpable breast masses. The real-time images were observed on a television monitor while moving the hand-held transducer probes over the masses. It took only a few minutes to examine and diagnose a palpable mass. Among 45 carcinomas, 39 lesions were correctly diagnosed, four lesions were not detected by ultrasound and two were misdiagnosed as fibroadenomas. On the other hand, ten benign lesions were falsely diagnosed as breast cancers: seven mastopathies (including four sclerosing adenosis), two fibroadenomas and one abscess. The sensitivity and specificity for diagnosing breast cancer were 0.87 and 0.90 respectively. Real-time sonography is a simple, time-saving and useful tool for examining palpable breast masses. However, it should be realised that some breast cancers are difficult to image and differentiate from benign lesions.

[Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes].

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.

[Clinical therapeutic effect of adoptive immunotherapy using IL-2-cultured autologous lymphocytes].

Our method of adoptive immunotherapy (AIT) using autologous IL-2-cultured lymphocytes differs from so-called LAK therapy in several points. We (1) obtain cultured lymphocytes from effusion lymphocytes (EL) or regional lymph-node lymphocytes (RLNL), when possible, rather than peripheral blood lymphocytes (PBL), (2) use crude IL-2 to induce T cell proliferation and to maintain killer activity, (3) use sonicated autologous tumor extract as antigen (Ag) to stimulate proliferation of cytotoxic T cells, and (4) pretreat the patients with local administration of OK-432 before AIT to induce effector cells that act synergistically with transferred killer cells. Surface marker analysis showed that OKT3, IL-2 receptor, Leu 2+15- cells were elevated while Leu 11a and Leu 3+8+ cells were decreased. Culture of RLNL augmented the expression of Leu 3+8- marker. Both of PBL and RLNL responded to Ag, and their auto-tumor killing activities were augmented in about half of the patients while rarely decrease by the addition of Ag. Response rates of patients with pleural effusion due to breast cancer and those with liver metastasis of breast cancer were 94% and 60%, respectively. Moreover, the survival was prolonged in the treated patients with pleural effusion or gastric cancer patients with peritoneal dissemination.

Selective accumulation of a new antineoplastic agent, K-18 (human immunoglobulin conjugated melphalan), in Ehrlich carcinoma transplanted in mice.

The absorption of K-18 (human IgG conjugated melphalan) through the intestine and its selective accumulation in the tumor were studied. After oral administration of 14C-melphalan and 14C-K-18 to ICR mice with subcutaneously transplanted Ehrlich carcinoma, K-18 was detected at the tumor site by autoradiogram after 48 hrs, but melphalan was not. Immunofluorescence study showed that orally administered K-18 was absorbed from the intestine via both the portal and the lymphatic route. Moreover, immunoperoxidase staining revealed K-18 in cytoplasm of tumor cells, but not in bone marrow cells. These results demonstrate the absorption of K-18 from the intestine after oral administration and its selective affinity for tumor tissue.

[Adrenal pheochromocytoma associated with renal cell carcinoma: report of two cases].

Two cases of adrenal pheochromocytoma associated with renal cell carcinoma are reported. The first case was in a 56-year-old woman who had been treated for hypertension. Computerized tomography (CT) scan revealed a right renal tumor and a right adrenal mass. Endocrinological examination and 131I-MIBG scintigraphy confirmed the diagnosis of pheochromocytoma. Right radical nephrectomy was performed under stable blood pressure. The second case was in a 45-year-old man who had been treated for gastric ulcers. CT scan incidentally revealed a right renal tumor and a left adrenal mass. He was normotensive and endocrinologically normal. Right radical nephrectomy and left adrenalectomy were performed, followed by corticosteroid supplementation. In both cases, histopathological diagnosis was renal cell carcinoma and adrenal pheochromocytoma. Both patients had no clinical evidence for von Hippel-Lindau disease such as tumorous lesions of the central nervous system, spinal cord and retina, and cystic lesions of the kidney and pancreas.

[Effect of combined immunotherapy using two different BRMs; OK-432 and IL-2-cultured lymphocytes].

We have developed an adoptive immunotherapy (AIT) system using syngeneic tumor-bearer-spleen cells cultured with interleukin-2 (IL-2) and soluble tumor extract. The therapeutic effect of the AIT was significantly augmented by in vivo local preadministration of a streptococcal preparation, OK-432. The previous report demonstrated a mechanism in which OK-432 augments the effect of AIT. That is, OK-432 induces IL-2 in vivo and prolongs the in vivo life span of cultured, IL-2-dependent lymphocytes (CL). This paper describes another mechanism, that is, the synergism between CL and OK-432-induced host lymphocytes. Fresh spleen cells (FSC) obtained from mice in complete remission after chemotherapy (cyclophosphamide, 100 mg/kg) showed a clear synergistic anti-tumor effect on CL, when both were injected i.p. into MOPC104E-bearing BALB/c mice which had been inoculated i.p. with 1 X 10(5) tumor cells 5 days previously. The effect was greatest when the FSC: CL ratio was 4:1, whereas the administration of either FSC or CL alone had little effect. The effector population of CL that exhibited synergism on FSC was Lyt 2+, cytotoxic T cells. FSC and CL both needed a tumor-specific combination. In addition, OK-432-induced tumor-infiltrating, intraperitoneal lymphocytes had a similar synergistic effect on CL as assessed by the transplantability of intraperitoneal cells. Probably because of this mechanism, OK-432 showed a much higher augmenting effect on AIT using CL than in vivo administration of IL-2. This therapy system using OK-432 and CL is a proper model which, through combined use of different, correlated BRMs, may bring a great effect whereas the effect of single BRM is weak.

[Immunotherapy of gastric cancer].

Two immunotherapeutic methods were developed as adjuvant therapy for gastrointestinal malignancies, one using oral administration of a streptococcal preparation, OK-432, and the other with adoptive immunotherapy (AIT), local transfer of IL2-cultured and autologous lymphocytes, combined with local preadministration of OK-432. Tsuchitani and Nio in our laboratory have revealed experimentally that oral OK-432 stimulates tumor-specific and non-specific immune mechanisms of gut-associated lymphoid tissues and inhibits the growth of some murine syngeneic tumors that were transplanted into cecal patches. The clinical efficacy of oral OK-432 on gastric cancer patients has been examined by a controlled randomized trial involving a total of about 1000 cases. Oral OK-432 (5KE, once a week) was revealed to be significantly effective in curatively resected group but not effective in non-curatively resected group. Ten consecutive patients with peritoneal dissemination of gastric cancer were treated with intraperitoneal OK-432 and AIT. AIT was performed by transferring autologous lymph node-lymphocytes cultured for 13 days with crude IL2 and tumor-extract. The survival of the treated patients was significantly longer (50% survival period exceeded 1 year) than that of historical control (95 cases, 50% survival period was 5 months). These immunotherapeutic methods are expected to be useful for multidisciplinary therapy of gastric cancer by a combination of each method with other therapeutic methods.

[Experimental study on the optimal treatment schedule for combination of BRM (immunostimulators, cultured killer cells or interleukin-2) and chemotherapy].

In the present study we tried to reevaluate the optimal combination timing in the experimental treatment with BRM and chemotherapeutic agents. BALB/c mice with advanced malignant ascites tumor (MOPC 104 E) were treated with cyclophosphamide (CPA, 2 mg/kg) and BRM such as immunostimulator (OK-432, Lentinan or Bestatin), interleukin-2 (IL 2) or cultured killer cells. The survival of mice was prolonged when immunostimulators were given before CPA. However, no combined effect was seen when immunostimulators were administered after CPA. Treatment with cultured killer cells and in vivo IL 2 after immunochemotherapy (immunostimulator followed by CPA) was the most effective protocol in which immunostimulator, chemotherapy, killer cells and IL 2 respectively seemed to induce, regulate, supplement and amplify anti-tumor effector cells.

[The therapeutic effects of OK-432 combined adoptive immunotherapy (AIT) against liver metastases of breast cancer].

We studied the effect of OK-432 combined AIT in 24 cases of liver metastases of breast cancer. Eleven of the 16 patients (69%) who received intraarterial transfer responded to this therapy. On the other hand, no patients responded to intravenous or intraportal transfer. The minimum cell number for a therapeutic response was 10(9) cells. Four patients had abscopal effects after therapy. The serum CEA level paralleled the therapeutic effects. There were no severe side effects accompanying this therapy. These results indicate that intra-arterial OK-432 combined AIT should be the first choice therapy against liver metastases of breast cancer.

[Experimental and clinical study of adoptive immunotherapy combined with preadministration of OK-432: a method to augment the therapeutic effect].

Our previous method of adoptive immunotherapy using IL2-cultured autologous lymphocytes consists of (1) in vitro sensitization by sonicated autologous tumor extract, (2) the induction and proliferation of active CTL by crude IL2, and (3) the preadministration of OK-432 for the augmentation of the therapeutic effect. Here we describe a new method to augment the therapeutic effect of OK432-combined AIT. In BALB/c mice with advanced malignant ascites (MOPC 104E), serial therapy with OK-432, cyclophosphamide and AIT significantly prolonged the survival compared with other therapeutic schedules through synergism between host's effector cells induced by immuno-chemotherapy and transferred killer cells. Many patients with advanced malignancies, for example, unresectable gastrointestinal cancer, locally advanced breast cancer or lung metastases of breast cancer, respond to such immuno-chemo-lymphocytotherapy, while previous OK432-combined AIT was effective only in malignant pleural effusion or metastatic liver tumor from breast cancer or peritoneal dissemination of gastric cancer.

[Factors linked to 5-year survival after hepatectomy for hepatocellular carcinoma: univariate and multivariate analyses of 312 patients].

The purpose of this study was to clarify the factors linked to 5-year survival after hepatectomy for hepatocellular carcinoma (HCC). Three hundreds and twelve patients who underwent hepatectomy for HCC between 1985 and 1994 were observed for at least 5 years. These patients were divided into 2 groups: 160 patients who died within 5 years after hepatectomy and 152 patients who did not die within 5 years. Statistical analysis by chi 2 test showed that the favorable factors linked to 5-year survival were (a) diabetes mellitus (-), (b) albumin > 3.7 g/dl, (c) hepaplastin test > 61%, (d) indocyanine green retention test at 15 minutes < 16%, (e) curative resection (+), (f) alpha-fetoprotein < or = 32 ng/ml, (g) portal vein invasion (-), (h) intrahepatic metastasis (-), and (i) stage I and II. Statistical analysis by stepwise regression test showed that the favorable factors linked to 5-year survival were (a) intrahepatic metastasis (-), (b) diabetes mellitus, (c) indocyanine green retention test at 15 minutes < 16%, (d) curative resection (+) and (e) alpha-fetoprotein < or = 32 ng/ml. When patients diagnosed with HCC, patients selection for hepatectomy should be done, based on the total estimation, such as tumor invasiveness, liver functions, and diabetes mellitus.