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Tumor-infiltrating lymphocytes consist predominantly of T cells, whereas B cells, plasma cells, and natural killer cells are observed with different degrees of frequency. We investigated the nature of tumor-infiltrating B lymphocytes (TIB) in human lung cancer. First, to examine the ability of immunogloblin production by TIB, cancer tissues were subcutaneously transplanted in severe combined immunodeficient mice, and the murine serum was examined for the concentration of human immunogloblin. Human IgG (huIgG) was detected in the serum of all 12 mice engrafted with lung cancer tissues. huIgM was almost undetectable. The levels of huIgG reached a peak approximately 6 weeks after engraftment and gradually decreased but were detectable until 20 weeks postengrafment. Serum from a large cell carcinoma-engrafted mouse reacted with a protein of 60 kDa derived from lung cancer cell lines (PC-9, Sq-1) and autologous tumor cells but did not react with cell lysates of normal lung tissue. Serum from an adenocarcinoma-engrafted mouse reacted with two proteins, 33 and 55 kDa, derived from lung cancer cell lines (PC-9, Sq-1, A549) and autologous tumor cells but did not react with the lysate of normal lung tissues. These results suggest that B cells infiltrating lung cancer tissues produce IgG that recognizes common tumor-specific antigen.

BACKGROUND The growth pattern of early gastric carcinoma, based on a volumetric analysis, reflects biologic characteristics of the tumor. The authors investigated the microvessel density (MVD), expression of vascular endothelial growth factor (VEGF), and growth patterns in early gastric carcinoma. METHODS Ninety-four tissue specimens resected from patients with early gastric carcinoma invading the submucosal layer were examined. Microvessel quantification was performed immunohistochemically using a monoclonal antibody against factor VIII-related antigen. VEGF expression was studied using an anti-VEGF polyclonal antibody. Growth patterns were defined as follows: Pen A type: expansively penetrating growth; Pen B type: infiltratively penetrating growth; Super type: superficially spreading growth. RESULTS The mean MVD was 16.9 (range, 5.2–43.0). MVD was significantly higher in tumors with venous invasion (P < 0.01), lymphatic vessel invasion (P < 0.05), and lymph node metastases (P < 0.05) compared with MVD in tumors without venous or lymphatic vessel invasion or lymph node metastases. The VEGF-positive rate of Pen A type tumors was 66.7% (18 of 27), that Pen B type was 10.0% (1 of 10), that of Super type was 19.4% (6 of 31), and that of the unclassified type was 15.4% (4 of 26). The VEGF-positive rate in patients with Pen A type tumors was significantly higher than that in patients with the other three growth patterns(P < 0.01). MVD in patients with Pen A type tumors (25.9 ± 9.2) was significantly higher than that in patients with Super type tumors (12.6 ± 5.4) (P < 0.01). Patients with Pen A type tumors had a poorer prognosis than patients whose tumors had other growth patterns (P < 0.05). According to multivariate analysis, VEGF expression and lymphatic vessel invasion were significant prognostic factors. CONCLUSIONS Pen A type gastric carcinoma tends to secrete VEGF, thus inducing tumor angiogenesis and resulting in venous invasion. Intensive follow-up is necessary for patients with Pen A type tumors, because this tumor type has a greater propensity for hematogenous metastasis. Cancer 1999;85:2340–6. © 1999 American Cancer Society.

Lung cancer-specific cytotoxic T lymphocytes (CTL) were induced hy repeated stimulations of regional lymph node lymphocytes (RLNI,) in lung cancer patients with either autologous or HLA-A-locus-matched tumor cells. To investigate the effect of interleukin-12 (IL-12), IL-12 was added during the stimulation of RLNL from HLA A24 / adenocarcinoma patients with either autologous tumor cells or HLA A24-positive adenocarcinoma cells (PC-9) in combination with, or instead of interleukin-2 (IL-2), and then the cytotoxic activity, cytokine production and populations of the lymphocyte subsets were examined. The addition of IL-12, or the substitution of IL-2 by IL-12 was found to enhance the cytotoxic activity and the cytokine production (IFN-γ, GM-CSF) of the CTL, as compared with IL-2 alone. The cytotoxic activity and cytokine production were both partially inhibited by anti-MHC-class I monoclonal antibody. The CTL thus induced by IL-12 had a higher proportion of CD3 + /CD56 + cells than the CTL induced with IL-2 alone. The positively selected CD5 + /CD56 - lymphocytes showed PC-9-specific cytotoxic activity, because the population did not show any cytotoxicity to K562 or A549 (HLA-A26/A30). However, the CD3 + /CD56 + lymphocytes were cytotoxic to both PC-9 and K562. In conclusion, IL-12 is considered to be a useful cytokine for both the induction of lung-cancer specific CTL and the augmentation of non-MHC-restricted cytotoxicity against tumor cells, and may he applicable for adoptive immunotherapy using CTL.

BACKGROUND The product of the p16INK4/CDKN2/MTS1 (p16) controls the transition from the G1 phase to the S-phase in the cell cycle by inhibiting the phosphorylation of the retinoblastoma gene product. A lack of p16 expression has been reported in various cancer cell lines and tumors; however, there have been only a few reports on the prognostic significance of p16 alteration. The authors studied p16 expression in nonsmall cell lung carcinoma (NSCLC) and also examined its correlation with clinicopathologic features and prognosis. METHODS p16 expression was determined by immunohistochemical analysis of 115 paraffin specimens of primary NSCLC that were curatively resected. The immunohistochemical study was performed using the labeled streptavidin-biotin method with anti-p16 rabbit polyclonal antibody. RESULTS Thirty-one of 115 NSCLC specimens (27%) showed negative p16 staining. The frequency of negative p16 expression was significantly higher in squamous cell carcinoma (39.5%) than in adenocarcinoma (20.3%) (P = 0.026). There were no statistically significant differences in the p16 status with respect to age, gender, smoking history, histologic differentiation, or stage of the disease. The Kaplan-Meier survival curves demonstrated that patients with negative p16 expression survived for a significantly shorter period of time than those with positive p16 expression (P = 0.043). p16 status was a significant prognostic factor, especially in patients with early stage disease (Stages I-II) (P = 0.039). CONCLUSIONS A lack of p16INK4 expression in NSCLC was observed more frequently in squamous cell carcinoma than in adenocarcinoma, and also was found to be closely related to prognosis, especially in patients with early stage squamous cell carcinoma. Cancer 1997; 80:389-95. © 1997 American Cancer Society.

We analysed data on 1117 patients with gastric cancer who were treated by curative resection. Attention was focused on invasion and a recurrence of the cancer. Based on a univariate analysis, death following a recurrence and prognosis were related to age of the patients, size of the tumour, tumour location, tumour tissue differentiation, growth pattern, depth of invasion, lymphatic and vascular invasion and lymph node metastasis. In proportion to the growth potential, determined by the level of proliferating cell nuclear antigen (PCNA) labelling, the death related to a recurrence was increased and the prognosis was poorer. Multivariate analysis showed that the three factors of serosal invasion, PCNA labelling index and lymph node dissection were independent prognostic factors. When sites of recurrence were analysed regarding each depth of invasion, haematogenous recurrence, in particular in the liver, occurred even in cases of an early invasion and many types of recurrences, including peritoneal recurrence, were noted in patients with an advanced state of invasion.

BACKGROUND Several studies have proven the usefulness of microvessel quantification as a prognostic factor for patients with various malignant tumors. The aim of this paper was to clarify the relationship between microvessel density (MVD) as a parameter of tumor angiogenesis and liver metastasis in colorectal cancer. METHODS A total of 175 patients with advanced colorectal cancer were evaluated (58 with concurrent liver metastasis). Microvessel quantification was performed immunohistochemically, using monoclonal antibodies against endothelial protein Factor VIII-related antigen (F8RA) and against endothelial surface marker CD34. Finally, the relationship between MVD and liver metastasis was analyzed. RESULTS A significant correlation was observed between MVD for F8RA and MVD for CD34 (n = 175, r = 0.9560, P = 0.0001). MVD in the tumors stained for F8RA ranged from 15.2 to 78.6 microvessels per × 200 field (mean 32.8 ± 11.7), while the tumors stained for CD34 varied between 21.6 and 118.8 microvessels per × 200 field (means 56.1 ± 20.5). A significantly higher MVD was observed in the tumors with liver metastatic disease compared with the tumors without liver metastasis (F8RA: mean 36.1 ± 11.3 vs. 31.2 ± 11.5, P = 0.0090; CD34: mean 64.4 ± 20.4 vs. 52.0 ± 19.4, P = 0.0010). CONCLUSIONS Microvessel quantification within a colorectal tumor using immunohistochemical staining methods has shown a significant correlation between MVD and liver metastasis. Tumors with a greater MVD may thus have a greater hematogenous metastatic propensity. Cancer 1996;77:1722-8.

BACKGROUND. We examined the relationship between the expression of a multidrug resistance-associated protein (MRP) and the biologic factors regarding invasion and metastasis of human gastric cancer. METHODS. In 75 patients with gastric cancer, the expression of MRP was immunohistochemically investigated and the expression of MRP mRNA was also detected using reverse transcription PCR (RT-PCR). Sensitivity to the anticancer agents, cisplatin (CDDP), doxorubicin (DXR), etoposide (VP-16), and mitomycin C (MMC) was examined using the MTT {3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl[2H]-tetrazolium bromide} assay. The relation between MRP expression and development, invasion, and metastasis of cancer was analyzed, and overexpression of the tumor suppressor gene p53 was investigated, immunoltistochemically. RESULTS. Immunohistochemically detected MRP positive tumors were noted in 34 of 75 excised tumors (45%), and confirmed by RT-PCR. There was no significant relation between MRP expression and clinicopathologic features or prognosis. Positive p53 staining was evident in 16 of 34 MRP positive tumors (47%) and 18 of 41 negative ones (44%), and there was no significant correlation between MRP and abnormal p53 expression. The MTT assay showed that MRP positive gastric cancer tissue was less sensitive to CDDP, DXR, and MMC compared with MRP negative ones. A similar tendency was noted with VP-16. CONCLUSIONS. MRP expression relates to the chemosensitivity of tumor cells against some anticancer drugs and is independent of known factors related to the development, invasion, and metastasis of human gastric cancers.

Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour. Images Figure 1

Combined aortoesophageal resection was performed in 8 patients, including 7 with esophageal carcinoma and 1 with aortoesophageal fistula. Aortic resection procedures included segmental resection with permanent aorto-aortic bypass (1 case), segmental resection with graft interposition (1 case), semicircumferential resection with patch aortoplasty (3 cases), wedge resection with lateral aortorr-haphy (1 case), and resection of adventitia (2 cases). Protective methods during aortic cross-clamping included one aorto-aortic permanent bypass, one subclavian-aortic bypass, and three axillo-femoral bypass. Postoperative complications include mediastinal abscess, paresis, arrythmia, and pneumonia. Five patients with esophageal carcinoma died within 6 postoperative months. In 4 of these 5 nonsurvivors, metastasis to distant organs including the liver, bone and peritoneal cavity were found at the time of death or autopsy. Those early recurrence cases were characterized by skip lesions and extensive lymph node metastasis with extranodal invasion. The clinical benefit of aortoesophageal resection will be attained by careful preoperative evaluation for case selection and a sufficient protective method for aortic cross-clamping.

BACKGROUND Many reports have concluded that quantification of the argyrophilic nucleolar organizer regions (AgNORs) measures proliferative activity and is a prognostic indicator in malignant disease. This retrospective study set out to evaluate the relationship between the AgNORs of the primary tumors and those of lymph node metastases in esophageal carcinoma. METHODS Using a one-step silver staining technique, AgNORs were counted in surgical specimens from 54 patients with squamous cell carcinomas. RESULTS The AgNOR scores of the lymph nodes metastases were significantly lower than those of the primary tumor (P = 0.0001). In 53 of 54 cases (98%), the AgNOR scores in the nodal metastases were lower than those of the primary tumor. The survival of 22 patients with AgNOR scores > or =4.0 for the primary tumor was significantly less than that of 32 patients with AgNOR scores

To evaluate the relation between the degree of lymph node metastasis and the growth potential of tumour cells and the local immune function in gastric cancer, we analyzed data on 444 patients with advanced serosally invasive gastric cancer who underwent curative gastrectomy. Tumour growth potential was evaluated based on the value proliferating cell nuclear antigen (PCNA) in the primary tumour, and dendritic cell infiltration into the tumour was determined as an indicator of local immune function. The values of PCNA labeling in the primary tumour increased and the infiltration of dendritic cells into the tumour decreased in relation to the extent of lymph node metastasis. High growth potential and low immune function were seen in cases with n3 lymph node metastasis. There was a reverse relation between the PCNA labeling index and dendritic cell infiltration. A variety of forms of recurrence was noted in patients with lymph node metastasis while the prognosis was less favorable, in relation to the degree of lymph node metastasis. Thus, the potential for nodal spread appears to be associated with the growth potential of tumour cells and with the local immune status of the tumour.

Evidence has accumulated that, in addition to the MDRI gene-coded P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) also mediates the multidrug resistance (MDR) of various human tumors. In the case of gastric cancer, there is little or no involvement of P-glycoprotein, and the mechanisms of MDR remain to be understood. To search for a possible relationship between expression of MRP and sensitivity to anti-cancer agents in gastric cancer, 4 gastric cancer cell lines, 43 human gastric carcinomas and 17 adjacent normal gastric tissue samples were analyzed. Expression of MRP mRNA was evaluated using reverse transcription PCR (RT-PCR) and Southern hybridization. Sensitivity of the test samples to the anti-cancer drugs cisplatin (CDDP), doxorubicin (DXR) and etoposide (VP-16) was examined using the MTT{3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl [2H]-tetrazolium bromide} assay. Immunohistochemical staining with the use of the MRP antibody (MRPrI) was done to confirm the findings regarding the expression of mRNA levels. The MRP expression evaluated with RT-PCR and Southern hybridization as well as with immunohistochemical staining revealed that 23 of 43 gastric-cancer tissues (53.5%), 15 of 17 normal gastric tissues (88%) and 3 of 4 gastric-cancer cell lines (75%) were positive. The MTT assay showed that DXR was significantly more sensitive (p < 0.01) in gastric carcinoma tissues lacking MRP expression than in those with positive expression. The same tendency was seen with the other agents used. Of the cell lines, one which showed no MRP expression also had a higher sensitivity to CDDP, DXR and VP-16 than the other positive cases. These results show that MRP expression is involved in MDR of human gastric cancer and is inversely related to the chemosensitivity of tumor cells against some anticancer drugs. © 1996 Wiley-Liss, Inc.

BACKGROUND The clinicopathologic features of young and elderly patients with gastric carcinoma have been analyzed. METHODS We analyzed the data from 174 patients with gastric carcinoma age 40 years and younger and from 356 patients with gastric carcinoma age 70 years and older who were surgically treated at the Department of Surgery II, Kyushu University, Japan. RESULTS The rate of multiple gastric carcinomas was 2.9% (5/174) for the young patients and 13.2% (47/356) for the elderly. In subjects older than 70 years, male patients predominated, tumors were smaller, differentiated lesions more common, vascular involvement more frequent, tumors were less infiltrative, and the rate of liver metastasis was higher. For patients younger than age 40 years, undifferentiated type with infiltrative growth was frequent and the rate of liver metastasis was higher. There were no differences in the positive rate of p53 overexpression and the proliferating activity of the cancer cells determined by PCNA LI, between the young and elderly patients. The survival rate after curative resection was lower for the elderly compared with that for the young patients; hematogenous recurrence was higher in the former. CONCLUSIONS The clinicopathological features of gastric carcinoma differed between the young and elderly patients, and these differences should be considered when age-oriented treatment is being designed. Cancer 1996;77:-1774-80.

Background. Even after curative resection of an early gastric cancer, some patients die of a recurrence. It is our view that patients with early gastric cancer who died of their disease had occult micrometastases in perigastric lymph nodes at the time of the original diagnosis. In an attempt to identify these micrometastases, lymph nodes dissected from early gastric cancer lesions were stained after operation with a monoclonal antibody against cytokeratin, an essential constituent of the cytokeleton of epithelial cells. Methods . The 420 dissected lymph nodes from 34 patients with node-negative early gastric cancer who died of a recurrence were examined for the presence of tumor cells. We used immunocytochemical techniques and an antiserum to epithelial membrane antigen. The monoclonal antibody CAM 5.2 recognizes cytokeratin polypeptides (human cytokeratin numbers 8 and 18) commonly present in epithelial cells. Clinicopathologic characteristics and prognosis were determined for patients with cytokeratin-positive cells in the lymph nodes. Results . Of 420 lymph nodes, 15 (3.6%) nodes and 23.5% (8 of 34) of the patients presented with cytokeratin-positive cells at the time of primary operation. The presence of cytokeratin positivity was not related to various clinicopathologic factors. The histologic stage of eight cytokeratin-positive cases was upstaged by the group of cytokeratin-positive lymph nodes from stage I to three of stage II, four of stage III, and one of stage IV, hematogenous recurrences were common, and the prognosis was poorer. Conclusions . Immunohistochemical techniques aid in identifying micrometastatic disease in lymph nodes missed in routine hematoxylin-eosin staining. Cytokeratin staining of the dissected lymph nodes is recommended to precisely determine tumor stage and prognosis for patients with early gastric cancer.

Data on 715 Japanese patients with gastric cancer were studied retrospectively with regard to the relationship between macroscopic and microscopic diagnoses of serosal invasion and clinicopathological factors affecting the accuracy of the macroscopic diagnosis. Although there was no macroscopic evidence of serosal invasion intraoperatively (S0 or S1), there was histological evidence of cancer cells on the serosal surface in 69 patients (9.7%). In these serosal invasion-positive cases, the tumors were larger ; were located more commonly in the upper third, lesser and greater curvatures of the stomach ; were Borrmann type 3 or type 4 tumors, and of an undifferentiated histologic type with an infiltrative growth pattern more commonly, and had more extensive lymphatic and vascular vessel invasion and lymph node metastasis (P < 0.01). Total gastrectomy was done more often for the serosal invasion-positive group, but the extent of lymph node dissection was comparable. Cases of a noncurative resection because of a positive surgical margin were more frequent in the serosal invasion-positive gorup (8/69 vs. 14/646, P < 0.01), and most had undifferentiated and infiltrative cancers. The 10-year survival rates were 49.2% and 85.5% for patients with and without serosa invasion, respectively. These findings clearly show that the serosal surface, especially in cases of the undifferentiated or infiltrative type of gastric cancer, must be closely inspected intraoperatively.

We treated two patients with a ruptured amebic liver abscess. The diagnosis was made at a relatively early stage and treatment was successful for one patient, but an accurate diagnosis of liver abscess was not made and invasive extraintestinal amebiasis led to multiple organ failure and to death for the other. Neither patient had been outside of Japan, and both were heterosexual. The origins of Entamoeba histolytica infection could not be determined. Though the mortality rate is high in cases of ruptured amebic liver abscess, appropriate management can lead to a good prognosis.

We detected two size classes in rat liver α2u-globulin mRNAs when analyzing cDNA clones, and named the mRNAs corresponding to the cDNA clones having longer and shorter sizes as L-type and S-type, respectively. When sequencing these cDNA clones, a 25 base insertion was present in the L-type at the 6th exon-intron junction. The extra sequence coincided with the 5′ part of the reported 6th intronic sequence that is directly contiguous to the 3′ part of the 6th exon. The relative abundance of L- and S-type mRNAs in the steady-state liver was determined by a sensitive S1 nuclease analysis using the probe prepared from the L-type cDNA. The same method was also applied for the determination of the L/S ratios during the course of postnatal development and estrogen treatment, all resulting in similar values, of about 0.03. These findings indicated that L-type and S-type mRNAs are generated by an alternative splicing mechanism in the 3′ noncoding region of α2u-globulin transcripts. Sequence analysis also elucidated the presence of at least two active genes showing exactly the same pattern of alternative splicing.

Background: Recently, some studies have focused on the tumor angiogenesis and its prognostic value. We studied the expression of vascular endothelial growth factor, microvessel counts, and serum concentrations of vascular endothelial growth factor to investigate their association with clinicopathologic factors and prognosis in non-small-cell lung cancer. Methods: The expression of vascular endothelial growth factor was determined by an immunohistochemical analysis from 91 paraffin specimens of completely resected non-small-cell lung cancers using anti–growth factor polyclonal antibody. Microvessel staining was performed by immunohistochemical analysis with anti–factor VIII–related antigen polyclonal antibody. Measurement of the serum concentrations of vascular endothelial growth factor used the sandwich enzyme-linked immunosorbent assay technique. Results: Expression of vascular endothelial growth factor was detected in 48 of the 91 tumors. The positive ratio was significantly higher in patients with adenocarcinoma than in those with squamous cell carcinoma. The microvessel counts were significantly higher in the patients with nodal metastasis than in those without nodal metastasis. The serum concentrations of vascular endothelial growth factor were also significantly higher in the patients with T3-4 disease than in those with T1-2 disease. The microvessel counts were closely associated with expression of vascular endothelial growth factor. The prognosis of patients with a positive growth factor ratio was significantly worse than that of the patients with a negative ratio ( p = 0.002), especially in squamous cell carcinoma. According to a multivariate analysis, only nodal status and expression of vascular endothelial growth factor were found to be independent prognostic factors. Conclusions: The expression of vascular endothelial growth factor was one of the most important prognostic factors in completely resected non-small-cell lung cancer, especially in squamous cell carcinoma. (J Thorac Cardiovasc Surg 1998;115:1007-14)