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Esophageal squamous cell carcinoma (ESCC) often recurs after chemoradiotherapy, and the prognosis of ESCC after chemoradiotherapy has not improved over the past few decades. The mutation process in chemoradiotherapy-resistant clones and the functional relevance of genetic alterations remain unclear. To address these problems, we performed whole-exome sequencing of 52 tumor samples from 33 patients with ESCC who received radiotherapy combined with 5-fluorouracil/platinum. In multiregion analyses of pretreatment and locally recurrent lesions from five cases, most driver gene-altered clones remained under chemoradiotherapy selection pressure, while few driver gene alterations were acquired at recurrence. The mutation signatures of recurrent ESCC, including increased deletion frequency and platinum dose-dependent base substitution signatures, were substantially different from those of primary ESCC and reflected the iatrogenic impacts of chemoradiotherapy. Single-region analysis of 28 pretreatment tumors indicated that focal copy-number gain at the MYC locus was significantly associated with poor progression-free survival and overall survival after chemoradiotherapy. MYC gain remained throughout the chemoradiotherapy course and potentially contributes to intrinsic resistance to chemoradiotherapy. Consistent with these findings, MYC copy number and mRNA and protein levels in ESCC cell lines correlated positively with resistance to radiotherapy, and MYC knockdown improved sensitivity to radiotherapy. Overall, these data characterize the clonal evolution process induced by chemoradiotherapy and clinically relevant associations for genetic alterations in ESCC. These findings increase our understanding of therapeutic resistance and support the rationale for precision chemoradiotherapy. SIGNIFICANCE: Whole-exome sequencing reveals the genetic evolution of ESCC during chemoradiotherapy, highlighting MYC gain in pretreatment tumors as a potential marker of therapy resistance., (©2021 American Association for Cancer Research.)

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial-mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38-3.40 and HR = 3.92; 95% CI = 2.27-6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50-11.57) and Dukes C (HR = 2.57; 95% CI = 1.42-4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value., (©2012 AACR.)

Radiotherapy is a useful component of treatment for esophageal cancer. Identification of the genes that are differentially expressed between radiosensitive and radioresistant cancer cells is important for predicting clinical effectiveness of radiotherapy. We established human esophageal cancer cell lines resistant to X-ray. Using differential display, we obtained one gene that was expressed in radiosensitive cells but was rarely expressed in radioresistant cells, and that gene was identical with hepatoma-derived growth factor (HDGF), an acidic polypeptide with mitogenic activity for fibroblasts. The semiquantitative reverse transcription-PCR assay confirmed that HDGF mRNA expression was reduced in established radioresistant cells, and its reduction was associated with reduced sensitivity to irradiation. Radiotherapy was more effective in clinical cases with high HDGF mRNA expression compared with cases with low expression (P < 0.05). The findings demonstrate that HDGF may play an important role in radiosensitivity, and it could be a novel marker predicting effectiveness of radiotherapy in clinical cases.

Applications of nonviral vectors for gene transfer into tumors in vivo have been limited by the relatively low expression levels of the transferred gene. The aim of this study is to evaluate the efficacy of electroporation-mediated interleukin-12 (IL-12) gene therapy for hepatocellular carcinoma (HCC). First, we investigated the optimal conditions of electric pulses (voltage, pulsing duration, numbers of shocks) of in vivo electroporation for gene transfer into HCC established by s.c. implantation of MH134 cells to C3H mice. This process made use of plasmid DNA that express the luciferase gene. We concluded that the optimal conditions for the electric pulses are as follows: voltage at 150 V; pulsing duration at 50 ms; nonpulsing duration at 950 ms; and the number of shocks at 10. Second, we tried to treat s.c. HCC by electroporation using plasmid DNA that expresses the murine interleukin-12 (mlL-12) gene. Intratumoral administration of the mIL-12 vector elevated serum IL-12 and IFN-gamma and significantly inhibited the growth not only of HCC into which the mIL-12 vector had been directly transferred, but also of the distant HCC. In addition, intratumoral administration of the mIL-12 vector inhibited spontaneous lung metastasis and delayed establishment of HCC injected 3 days after mIL-12 gene therapy. The IL-12 gene therapy induced more lymphocyte infiltration by NK cells, CD3+ cells, and Mac-1 positive cells into the tumor and reduced the number of microvessels. Therefore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive tumor cells were found. These results demonstrate that gene therapy for HCC by electroporation in vivo using IL-12 is very efficient and is thus promising for further clinical trial.

Human papillomavirus type 16/18 (HPV-16/18) is implicated in the pathogenesis of squamous cell carcinoma (SCC) of the cervix and esophagus. The arginine allele at codon 72 of p53 was found to be more susceptible to degradation by HPV E6 protein than is the proline allele in vivo, thus resulting in a high frequency of cervical SCC in individuals homozygous for arginine at the codon. There are controversial results from several clinical studies of cervical SCC. In the present study, encoding regions of p53 codon 72 and HPV-16/18 E6 were directly sequenced, using pairs of primary esophageal SCC tissue and corresponding normal mucosa, which were from 75 patients (Japanese, n = 38; Chinese, n = 37). The arginine allele alone was detected in 70.6% (12 of 17) of HPV-positive cases but only in 43.1% (25 of 58) of HPV-negative cases (P < 0.05). In contrast, such a significant correlation between p53 polymorphism and HPV infection was not evident in corresponding normal mucosae. Because our findings between tumor specimens and the normal mucosae differed, we suggest that the frequent loss of proline allele in HPV-associated carcinogenesis of the esophagus major plays some role. The particular type of p53 polymorphism may indicate a potential candidate for HPV-associated SCC.

Interleukin-6 (IL-6) was found to function as a late-acting killer helper factor in the differentiation of CTLs. In the model of tumor-bearing mice, the systemic administration of recombinant IL-6 was found to mediate the antitumor effect on the immunogenic murine tumors via the in vivo induction of murine CTLs but not on the poorly immunogenic murine tumors in our previous study. However, an in vivo experimental model capable of analyzing the anti-human tumor effect via the in vivo induction of human CTLs has not yet been established. Therefore, in the present study, severe combined immunodeficient mice were given human peripheral blood lymphocytes (SCID-PBL/hu), and thereafter human tumor cells were administered i.p. into these SCID-PBL/hu mice as a model of human patients with cancer. When these SCID-PBL/hu mice bearing allogeneic human CESS B blastoid tumor cells were treated in vivo with recombinant adenovirus vector expressing IL-6 cDNA, both the induction of CD8+ human CTLs against CESS cells in the spleen cells and peritoneal exudate cells and a prolongation in the survival of these mice were observed. Furthermore, SCID-PBL/hu mice were given peripheral blood lymphocytes from patients with cancer (gastric or rectal cancers) and autologous human tumor cells. The in vivo administration of recombinant adenovirus vector expressing IL-6 cDNA induced CD8+ human CTLs specific for autologous human tumor cells from human precursor T cells. The in vivo injection of the IL-6 gene also inhibited growth and metastasis in autologous human cancers. Based on the above findings, the experimental model using SCID-PBL/hu mice and the IL-6 gene delivered in vivo by an adenovirus vector might therefore provide a new strategy capable of analyzing an anti-human tumor effect and the in vivo induction of human CTLs by cytokine gene therapy without using the human body.

Growth factor receptors transmit intracellular signals that may be important in carcinogenesis. The Grb7 protein was recently identified as a substrate of the epidermal growth factor receptor and related Her2/ erbB2 receptor-linked tyrosine kinase activity. The Grb7 gene has been found to be coamplified with Her2/erbB2 in breast carcinomas. In this study, Grb7 expression was studied in 32 human esophageal cancers. A human Grb7 cDNA encoding for N-terminal amino acids was isolated and found to be 90% homologous to the murine counterpart. Although there was no amplification of the Grb7 gene in esophageal cancers, Grb7 mRNA was found to be overexpressed in 14 cancers (43.8%) but not in adjacent normal esophageal mucosa. It is noteworthy that coexpression of Grb7 with epidermal growth factor receptor or Her2/erbB2 was detected in 10 esophageal carcinomas (31.3%) and was significantly related to extramucosal tumor invasion (P = 0.02), whereas such a relationship was not shown by each sole expression. These findings suggest a possible relationship of Grb7 signaling in association with expression of tyrosine kinase receptors in aggressive human esophageal cancer.

Thioredoxin (TRX), a disulfide-reducing intracellular protein, functions as a cellular defense mechanism against oxidative stress. In this study, we asked whether expression of TRX, glutathione-thiol transferase pi, and high mobility group protein 1 (HMG-1) genes is enhanced in human hepatocellular carcinoma and whether expression of these genes is associated with sensitivity to cisplatin. Both TRX and HMG-1 were co-overexpressed in almost all cancerous lesions in comparison to normal tissue in surgically resected hepatocellular carcinomas of 20 patients. Tumor sensitivity to cisplatin [cis-diamminedichloroplatinum (II)], but not to mitomycin C or doxorubicin, correlated with mRNA levels of TRX in cancer tissue. TRX and HMG-1 may be useful tumor markers, and TRX might be also a useful marker for sensitivity to cisplatin in human hepatocellular carcinomas.

Analyzing microsatellite instability (MI) in malignant tumors is thought to be useful for screening cancer patients to identify those patients with a higher risk of developing second malignant tumors. In this paper, we report a new, accurate, and efficient method of detecting MI using an automated fluorescent DNA sequencer and a computer that automatically calculates the size, height, and area of each fluorescent product, making it possible to assess MI more accurately and more rapidly. The primers for amplification of each microsatellite locus are labeled by two different fluorescent dyes, rox (red) and fam (blue). The rox-labeled primer was used for the tumor, whereas the fam-labeled primer was used for the corresponding normal tissue. Two amplified products from both the tumor and the normal tissue were co-loaded into a single lane of the sequencing gel and were analyzed. MI could be detected based on the presence of different waving patterns. Furthermore, several loci could also be analyzed simultaneously for MI in a single lane. Using this method, we examined the frequency of MI in gastric cancer. The results showed that 5 of 22 (22.7 %) gastric cancers were MI-positive, which corresponds to the findings of previous reports that used the radioisotopic method. The improved method may open up the possibility of performing routine examination of MI in many cancer patients and offers hope for the potential clinical application of Ml analysis as a follow-up evaluation of cancer patients.

We examined, immunohistochemically, tissues from primary lung adenocarcinomas. In 142 tumors, the mean overall labeling percentage of cathepsin B was 26.5 +/- 22.3 (SD). The mean labeling percentage of cathepsin B in cases with stage I disease was lower than that in cases with stages IIIA, IIIB, or IV disease (P < 0.05). Of the 115 tumors examined for laminin-positive basement membranes, 54 (47%) had a continuous pattern and 61 (53%) had a discontinuous pattern. The mean labeling percentage of cathepsin B was 35.0 +/- 24.2 in tumors with a discontinuous pattern, compared with the 21.9 +/- 16.9 in those with a continuous pattern (P < 0.01). The overall 5-year survival rates of patients with high and low cathepsin B expressions were 26% and 77%, respectively (P < 0.01), including 45% and 94% for patients with stage I disease, respectively (P < 0.01), and 15% and 60% for those with stage IIIB disease, respectively (P < 0.05). Multivariate analysis using the Cox life table regression model showed cathepsin B to be a significantly independent factor associated with death due to the disease. We conclude from this study that tumors with a discontinuous pattern of laminin have a higher percentage of cathepsin B, and the survival rate was poor for patients with a high expression of cathepsin B. Thus, cathepsin B may be useful in assessing prognosis in lung adenocarcinoma.

Cell proliferation in 211 primary non-small cell lung carcinomas was assessed by using an immunostaining of proliferating cell nuclear antigen (PCNA) and a silver staining of argyrophilic nucleolar organizer region (Ag-NOR). More than 5% PCNA positive cells was designated PCNA(+), and less than 5% was PCNA(-). A mean number or more of the Ag-NOR was a high Ag-NOR count, and less than the mean number was a low Ag-NOR count. The proportion of the tumors with PCNA(+) and high Ag-NOR counts showed an increase in patients in an advanced stage of the disease (P < 0.05). In 125 patients with stage I disease, the 5-year survival rate was 20% in patients with PCNA(+) and high Ag-NOR counts, compared with 47% in those with either PCNA(+) or high Ag-NOR counts and 76% in those with PCNA(-) and low Ag-NOR counts (P < 0.05). Our data suggest that the tumors with PCNA(+) and high Ag-NOR counts have a high proliferative activity. The combination analysis of PCNA and Ag-NOR may be useful in assessing prognosis in lung cancer, even in stage I disease.

Human hepatocellular carcinoma (HCC) often contains intratumoral subpopulations of heterogeneous cellular differentiations within each tumor. To analyze the genetic alterations of p53 in the heterogeneous subpopulations, we examined 68 intratumoral nodular lesions within 34 HCCs composed of two distinct subpopulations. The cellular differentiations were determined histologically by Edmondson's grading system. Nine (26.5%) of 34 HCCs examined were found to have genetic alterations in exons 5 to 8 of the p53 gene, resulting in amino acid substitutions. Three of these nine HCCs with p53 mutations showed genetic heterogeneity of the p53 gene within each tumor; one HCC had a single missense mutation at codon 210 (asparginine to 210-serine) in an intratumoral lesion of Edmondson Grade II and double missense mutations at codons 210 and 217 (asparginine to 210-serine and valine to 217-alanine) in another intratumoral lesion of Edmondson Grade III. The remaining two HCCs had p53 mutations only in lesions of a higher grade. In total, the p53 mutations were detected in none of eight Edmondson Grade I lesions, in five of 29 Grade III lesions (17.2%), in eight of 26 Grade III lesions (30.8%), and in three of five Grade IV lesions (60.0%). Thus, our data revealed that the p53 mutations were closely related to the progression of HCC and that, in certain cases, malignant cells which acquired the p53 mutations might develop into dedifferentiated subpopulations within individual HCC.

Two monoclonal antibodies, TKH2 and B72.3, directed toward the Sialyl-Tn antigen (SA 2, 6GalNAc alpha-O-Ser/Thr), were examined immunohistochemically to analyze the expression of these antigens in 20 areas of normal squamous epithelium, 12 lesions of dysplasia, and 86 cases of squamous cell carcinoma including 32 with superficial carcinoma in the esophagus. No expression of TKH2 or B72.3 was found in the normal squamous epithelium. Among the 12 lesions of dysplasia only one expressed TKH2. In carcinoma the expression of TKH2 and B72.3 was found in 40 (47%) and 21 (24%) of the 86 carcinomas, respectively; however, the number of positive malignant cells with TKH2 and B72.3 totaled less than half that in the tissue, and no relationship was found between either prognosis or lymph node metastasis and the expression of Sialyl-Tn antigen. These results indicate that Sialyl-Tn antigen appears in the process of malignant transformation or tumor progression in esophageal squamous cell carcinoma; however, the positive expression of Sialyl-Tn antigen was not directly connected to either prognosis or lymph node metastasis.

The combined effects of flavone acetic acid (FAA), a synthetic flavonoid, and hyperthermia on B16 melanoma cells were investigated. In vitro, FAA alone at concentrations below 100 micrograms/ml was not cytotoxic with a 60-min exposure at 37 degrees C. Hyperthermia at 43 degrees C for 60 min enhanced the cytotoxicity of FAA only at concentrations over 100 micrograms/ml. Inhibition of the growth of B16 melanoma solid tumor by FAA and/or hyperthermia was examined in vivo. FAA (100-200 mg/kg) inhibited tumor growth in a dose-dependent manner. The combined treatment of FAA (200 mg/kg) and hyperthermia (43 degrees C, 15 min) significantly inhibited tumor growth compared to a treatment of FAA or hyperthermia alone. The maximum antitumor effect of FAA combined with hperthermia was obtained when FAA was administered 2 or 4 h before heat. The significantly increased cytotoxicity of FAA combined with hyperthermia seems to relate to specific decreases in tumor blood flow, a reduction in tumor pH, and an increased tumor temperature, without altering pH in the normal tissues. This combined treatment of FAA and hyperthermia warrants further study for treating subjects with solid tumors.

Adenocarcinoma of the lung obtained at surgical resection was examined for mutation at codons 12, 13, and 61 of the oncogenes K-ras, H-ras, and N-ras, using polymerase chain reaction and oligonucleotide hybridization techniques. The mutation was detected in 18 of the 115 cases (15.7%), and 15 of 18 were at codon 12, 2 were at codon 13 of K-ras, and 1 was at codon 61 of N-ras. G to T transversions were most common. The ras gene mutations were more frequent in the male patients (P = 0.0048). No significant differences were found to be related to stage of the disease or tumor-nodes-metastases classification between positive and negative groups of the ras gene mutations. A history of tobacco use was not always a factor contributing to mutation. Of the completely resected group without lymph node metastasis, the 5-year survival rate in the ras-positive group was 53.3%, which was significantly poorer than the 83.6% survival rate in the ras-negative group (P less than 0.05). Our findings suggest that ras gene mutations may be prognostic, especially in the early stage adenocarcinoma of the lung.

The purpose of this study was to elucidate a possible immune response to tumor cells mediated by tumor-infiltrating lymphocytes (TIL) in lung cancer. In flow cytometry, the majority of T-cells of TIL were CD45RA-, CD45RO+, and CDw29high, and expressed HLA-DR. The expression of interleukin 2 receptor beta chain increased in both CD4+ and CD8+ TIL compared with both types of T-cells in peripheral blood. These results indicate that the major population of TIL is activated memory T-cells. The TIL preparation, which was usually contaminated with 5 to 10% tumor cells, did not exhibit any response in autologous mixed lymphocyte-tumor culture even in the presence of interleukin 2 (IL-2) in all five cases tested. Although purified T-cells from TIL showed the positive response in only 1 of 10 cases tested without addition of IL-2, it occurred in 7 of 10 cases in the addition of a low concentration of IL-2. The IL-2-dependent response to irradiated autologous tumor cells was suppressed when nonirradiated autologous tumor cells were added to the culture. Culture supernatants of four lung cancer cell lines and freshly prepared lung cancer cells obtained from 6 cases exhibited suppressive activity against anti-CD3 antibody-induced mitogenesis of peripheral blood mononuclear cells from healthy donors. We suggest that, taken together, (a) the major population of TIL in lung cancer are activated memory T-cells, and they include tumor-reactive ones, and that (b) the function of the TIL is impaired by unavailability of IL-2 and/or by suppression due to lung cancer cell-derived factor(s).

C57BL/6 mice inoculated i.p. with 3LL tumor cells were treated by local combination therapy with Nocardia rubra cell wall skeleton (N-CWS) and recombinant interleukin 2 (rIL-2). The combination treatment significantly prolonged their survival and augmented lymphokine-activated killer (LAK) activity of peritoneal cavity lymphocytes (PCL), compared with treatments with rIL-2 alone or N-CWS alone. After in vitro culture of peritoneal exudate mononuclear cells with rIL-2, the nonadherent population derived from N-CWS-injected tumor-bearing mice showed a significantly higher LAK activity than did that population derived from saline solution-injected mice. When N-CWS-induced PCL were cocultured with either N-CWS-induced macrophages or control macrophages in the presence of rIL-2, their LAK activity was higher than that of control PCL. Therefore, it was suggested that N-CWS-induced PCL themselves have a more potent ability as precursors of LAK cells. Phenotypic analysis on PCL populations revealed that N-CWS-induced PCL contained increased proportions of CD3+CD4-CD8- cells and asialo GM-1+ cells compared with control PCL. These results suggest that N-CWS selectively accumulates potent LAK precursors, namely, CD3+CD4-CD8- T-cells and asialo GM-1+ natural killer cells, at the injection site and that LAK cells are efficiently induced by subsequent administration of rIL-2.

The argyrophilic nucleolar organizer region (AgNOR) of 100 cancer cells from biopsy specimens of esophageal squamous cell carcinomas in 98 surgically treated cases was examined, using a silver colloid staining technique on biopsy specimens. The number of AgNOR per nucleus (AgNOR number) was higher in the more advanced groups with regard to the length of the tumor (P less than 0.01), the depth of penetration (P less than 0.05), and lymph node metastasis (P less than 0.01). The survival of the patients with a high AgNOR number (greater than or equal to 6) was significantly poorer than those with either a medium range AgNOR number (4 less than or equal to-less than 6) (P less than 0.05) or a low AgNOR number (less than 4) (P less than 0.01). In the multivariate analysis including conventional clinicopathological factors, the AgNOR number was found to be one of the independent and significant variables (P less than 0.01). Because the AgNOR method is simple and can be applied to paraffin-embedded sections, the AgNOR number may provide potential benefit in the pretherapeutic assessment of malignant potentiality in esophageal carcinoma.

When the number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in 274 patients with non-small cell lung cancer, the mean number per nucleus in patients overall was 5.07 +/- 1.92 (SD). With the use of the tumor (T)-nodes (N)-metastasis (M) classification, the mean Ag-NOR count for patients with T1 and T2 disease was statistically lower than that for those with T3 and T4 disease (P less than 0.01). The mean Ag-NOR counts were lower in patients with N0 disease than in those with N1 and N2 disease (P less than 0.01); lower in patients with stage I disease than in those with stage II, IIIA, IIIB, or IV disease (P less than 0.01); and lower in patients with adenocarcinoma than in those with squamous cell carcinoma (P less than 0.01) or large-cell carcinoma (P less than 0.05). In 131 patients with stage I disease, the mean Ag-NOR count was 3.80 +/- 1.32, and the 5-year survival rates of patients with Ag-NOR counts of less than 3.80 and greater than or equal to 3.80 were 78 and 44%, respectively, including 78% and 25% for adenocarcinoma, respectively (P less than 0.01). However, there was no statistically significant difference for those in stage II, IIIA, IIIB, or IV, and in stage I (without an adenocarcinoma). Because patients with stage I non-small cell lung cancer and a high number of Ag-NORs had a poor prognosis, Ag-NORs can serve as a pertinent marker of an early recurrence.

The urokinase-type plasminogen activator (u-PA) was used to study 96 cases of lung adenocarcinoma and 49 cases of lymph node metastatic adenocarcinoma. We made use of the immunohistochemistry of paraffinized samples. u-PA was detected in the cytoplasm of tumor cells, and the number of positive cells was higher in patients with T2 or T3 than in those with T1 disease (P less than 0.01). These u-PA-positive tumor cells were more frequent in patients with N2 than in those with N1 disease (P less than 0.01). Such cells were also detected in patients with N1 rather than N0 disease (P less than 0.01). When we compared the frequency of u-PA-positive tumor cells in metastatic lesions with that in primary ones, the former tended to be higher. On the other hand, the frequency of u-PA-positive cells in primary tumors of patients with a recurrence was higher than in those with no recurrence. Thus, u-PA is an important prognostic indicator associated with tumor growth and lymph node spread. If u-PA is detected in a tumor, a recurrence can be expected.

Proliferative activities in 91 primary gastric carcinomas and 36 corresponding metastatic perigastric lymph nodes were investigated using Ki-67 labeling percentage and an argyrophilic nucleolar organizer region (AgNOR) count. Tumors with a high proliferative activity often metastasized to lymph nodes, and the proliferative activities of the primary lesion and the perigastric lymph node metastases were similar. A significant correlation was recognized between the Ki-67 labeling percentage and the AgNOR count (r = 0.744; P less than 0.001). The Ki-67 labeling percentage and AgNOR count proved to be useful predictors of nodal metastasis regardless of tumor size, depth of invasion, and histological type. Even when tumors are smaller (less than 7 cm) or the stage of the disease is early (pT1, 2), the formation of metastasis increased with an increased Ki-67 labeling percentage or AgNOR count. The combination analysis of depth of invasion with Ki-67 labeling percentage or AgNOR count gives a more precise prediction of nodal metastasis, compared with histological analysis alone.

We conducted case-control studies of hepatocellular carcinoma (HCC) and liver cirrhosis (LC) in relation to hepatitis C virus (HCV) and hepatitis B virus infection, involving 91 patients with HCC, 75 patients with LC who had no evidence of HCC, and 410 control subjects from the Japanese population. Serum antibody to HCV (anti-HCV) was detected by both enzyme-linked immunosorbent assay and recombinant immunoblot assay in 51, 51, and 3% of HCC, LC, and controls, respectively, whereas the corresponding prevalence of serum hepatitis B surface antigen (HBsAg) was 21, 11, and 2%, respectively. The relative risks (and 95% confidence intervals) for the presence of serum anti-HCV were estimated as 52.3 (23.9-114.3) for HCC and 64.4 (27.4-151.4) for LC. These values exceeded the relative risk of HCC (15.3) and that of LC (6.1) for positive serum HBsAg. Among male patients with HCC or LC, anti-HCV rates were very high in blood recipients (about 70%), heavy drinkers (46-62%), and those who had no identifiable risk factors (65-75%), indicating possible transmission of HCV via routes other than transfusion. No significant difference in anti-HCV status was observed between the HCC and LC groups. It was notable that anti-HCV was much less prevalent among HBsAg-positive patients with HCC or LC than among HBsAg-negative ones. There was a slight to moderate increase in HCC or LC risk among blood recipients and heavy drinkers after adjustment for anti-HCV status. These results indicate that, in Japan, the possible role of HCV infection in the etiology of HCC and LC is extremely large and seems to be more important than chronic hepatitis B virus infection.

In this study, we investigated the cytolytic activity of peripheral blood T-cells (PBT) obtained from nine patients with primary lung cancer treated by surgical adjuvant adoptive immunotherapy (AIT) with lymphokine-activated killer cells and low-dose recombinant interleukin 2 at the time of rebound lymphocytosis (24-48 h after AIT). In eight of nine patients, nonspecific cytotoxicity of peripheral blood lymphocytes significantly increased as compared with that of pre-AIT peripheral blood lymphocytes. However, purified PBT showed much less activity to kill tumor cells although they increased in number and were activated well in terms of increases in the expression of HLA-DR and interleukin 2 receptor. The cytolytic activity of post-AIT PBT was significantly enhanced when they were targeted to Fc receptor-bearing tumor cells (K562 or Daudi) with anti-CD3 (NU-T3) or anti-T-cell receptor (TCR)alpha beta (WT31) monoclonal antibody in all five patients examined. Phenotypically, the targeted cytotoxicity was predominantly mediated by CD8+ cells. The results indicated that in vivo-activated PBT by AIT could not exhibit direct cytotoxicity, but they acquired cytolytic potential, the effect of which was expressed by targeting to tumor cells.

In order to accurately determine sex hormone dependency and hormone responsiveness in human esophageal carcinoma, the effects of sex hormones on the growth of esophageal carcinoma cell lines, KSE-1 and KSE-2 cells, were examined in vitro and in vivo. Cell proliferation of cultured KSE-1 cells was inhibited by treatment of estradiol and enhanced by dihydrotestosterone (DHT), whereas KSE-2 cells were unaffected by these sex hormones. The heterotransplanted tumors of KSE-1 cells in nude mice possessed estrogen receptor (ER) and androgen receptor (AR), while the tumors of the KSE-2 cells had neither ER nor AR. When the tumor growth rates and serum hormone levels were monitored during the continuous administration of either estradiol or DHT, no significant differences were observed in either the serum hormone levels or tumor growth rates between male and female mice. The administration of estradiol significantly inhibited the growth of ER-positive and AR-positive KSE-1 tumors in both males and females in conjunction with an increase in the estradiol levels and a decrease in the DHT levels in the serum. However, the growth of ER-negative and AR-negative KSE-2 tumors was not influenced by either estradiol or DHT administration. These results suggest that the in vivo growth of human esophageal carcinoma cells with sex hormone receptor is influenced by circulating hormone levels and can be manipulated by systemic estradiol administration.

We immunohistochemically examined 131 primary human lung adenocarcinomas for the possible presence of autocrine factors. Transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) were considered growth factors with epidermal growth factor receptor (EGFR) as the receptor. Of these tumors, 87 (66%) showed a high expression of TGF alpha, 66 (50%) showed a high expression of EGF, and 55 (42%) were positive for EGFR reactivity. In the EGFR-positive cases, the 5-year survival rates of patients with high TGF alpha and low TGF alpha were 36% and 85%, respectively (P less than 0.05). The 5-year survival rates of patients with high EGF and low EGF were 25% and 77%, respectively (P less than 0.05). In contrast, in the EGFR-negative cases, there was no statistical difference between the 5-year survival rates of patients with either high TGF alpha or EGF and low TGF alpha or EGF. Because autocrine growth mechanisms are present in adenocarcinoma of the human lung, these events may contribute to clarification of tumor development, and perhaps even to a better prognosis.

Serum carcinoembryonic antigen (CEA) levels were determined serially in 30 preoperative and postoperative patients with differentiated and 47 with undifferentiated gastric cancers. Macroscopic noncurative resection of the stomach was done for those patients. There was no difference between survival curves in the differentiated and undifferentiated cases, and the 50% survival was 13.1 months for the differentiated group and 12.5 months for the undifferentiated group. Preoperative serum CEA levels were 10.4 +/- 5.2 ng/ml for the differentiated and 4.0 +/- 1.6 ng/ml for the undifferentiated cases, and CEA-positive rates were 20.0% for the differentiated and 14.9% for the undifferentiated cases. There was no difference in preoperative CEA values with regard to tissue types. In the course of tumor progression, CEA levels increased during the first postoperative year in the differentiated cases and related reciprocally to decreases in survival rates. Little change was noted in the undifferentiated cases. Therefore, the serial postoperative assay of serum CEA levels has predictability with regard to tumor progression in patients with a differentiated gastric cancer.

We have attempted to develop an in vitro assay system for predicting estradiol sensitivity of clinical cancer cells by measuring the effects of estradiol on the net DNA synthesis of primary culture cells. Superinoculation of neoplastic and normal cells onto confluent monolayers of a contact-sensitive cell line, which have been designated as contact-sensitive plates (CSPs), resulted in both the specific growth of the neoplastic cells and the growth inhibition of contact-sensitive normal cells. The applicability of CSPs to an estradiol sensitivity test was examined in the known estradiol-sensitive, estrogen receptor-possessing cell lines, MCF-7 breast cancer and KSE-1 esophageal cancer cells. The responses of each cancer cell to estradiol were sufficiently evaluated in this assay and clearly demonstrated stimulative and inhibitive growth regulatory effects of the estradiol in MCF-7 and KSE-1 cells, respectively. A total of 38 clinical carcinomas (33 of the breast and 5 of the esophagus) were tested for their estradiol sensitivity. A statistically significant increase of cancer cell growth (P less than 0.1) in nontreated culture from the 48th to the 96th h of the primary culture on CSPs was observed in 28 of 38 overall cases (73.7%), and the evaluable data were obtained within 5 days by a sampling of 5 X 10(3) cancer cells. Most of the breast carcinomas exhibited a positive correlation between the growth-stimulative effect of estradiol in this assay and the estrogen receptor levels in the resected specimens. On the other hand, a clinical case of esophageal cancer with an estrogen receptor showed a growth inhibition of primary carcinoma cells by estradiol treatment. These results therefore indicate the feasibility of predicting individual tumor response to estradiol by using a rapid sensitivity test in vitro.

The role of microcirculatory factors within and around liver tumors after heat treatment was investigated in a rabbit model of liver cancer (VX2 carcinoma). As a physiological factor of the microenvironment, regional blood flow (RBF) was measured by the hydrogen clearance method, and a histopathological study was done. Local hyperthermia was administered directly to the liver tumor via a 915-MHz microwave. Hyperthermia produced a temporary reduction of RBF in both the tumor and the surrounding normal liver tissues, and the histopathology revealed congestion, petechiae, and thrombosis. After hyperthermia at 43.0 degrees C for 20 min, RBF in the tumor rapidly decreased to a minimum of 40% of the control level during 0-12 h after the treatment and increased gradually to the pretreatment level at 2 days. RBF in the normal liver also decreased rapidly after hyperthermia, to a minimum of 30% of the pretreatment level, 1-12 h after treatment. In the case of treatment at 42.5 degrees C for 20 min, RBF in the tumor also rapidly decreased to a minimum of 40% of the control level, at 4 h after the treatment, and recovery was within 2 days. However, RBF in the surrounding normal liver decreased to 80% of the control level, at 2 h after the treatment, and then increased more rapidly to reach levels seen in the controls. Thus, the latter condition of heat treatment was considered to be favorable for therapeutic gain. Based on the results of these sequential microcirculatory changes, the effects of continuous and intermittent hyperthermia were studied in groups given various treatments. In a group treated with intermittent hyperthermia at 4-h intervals, the antitumor effects determined by tumor growth retardation were significantly greater, as compared with findings in the group given treatment without an interval and that given at a 24-h interval (P less than 0.001). In the tumor at 4 h after hyperthermia, the increased thermosensitivity was considered to surpass the developing thermotolerance. Thus, the antitumor effect of hyperthermia in vivo greatly depends on the microcirculation. The most efficacious mode for application of hyperthermia must be vigorously examined if a clinical relevance is to be gained.

Eleven patients with gastric carcinoma, who had undergone re-resection of the lesion due to recurrence in the remnant stomach, were the subjects of a cytophotometric DNA analytical study. The objective was to determine whether or not the DNA cytogenic profile of cancer cells would be consistent during the growth of the carcinoma. The DNA distribution patterns were grouped into types I, II, and III, according to the proportion of aneuploid cell population. Ten of 11 had the same DNA distribution patterns in the primary and recurrent lesions, while in the other one patient with type III in the primary lesion, type II was evident at the time of recurrence. The DNA cytogenic profile of cancer cells is thus a valid cell marker of a given tumor and should be applicable for determining the natural history of gastric carcinoma.

The purpose of this study is to clarify the significance of antitumor activity of alveolar macrophages (AM) in lung cancer patients. AM from tumor-bearing and non-tumor-bearing segments were obtained separately by the lavage of bronchoalveolar tracts of resected lungs of 74 patients with primary lung cancer. Cytostatic activity (CTS) of AM obtained from non-tumor-bearing segments was stable in spite of enlargement of tumor size or progression of N factor. In contrast, CTS of AM obtained from tumor-bearing segments may be augmented at Stage II as compared with Stage I, and suppressed with an advance of stage from II through IV, although the number of Stage II patients was as small as three. Moreover, CTS of AM from tumor-bearing segments was suppressed in N2 as compared with N0 or N1, and also it was suppressed as compared with that of AM from non-tumor-bearing segments in N2 disease. CTS of AM from smokers was suppressed as compared with that of nonsmokers in both tumor-bearing and non-tumor-bearing segments. These results suggest that lung cancer cells or their products may suppress antitumor activity of AM in the tumor-bearing segments at advanced stages, and cigarette smoking is a suppressive factor on antitumor activity of AM.

We studied the effects of glucocorticoid replacement on tumor growth after adrenalectomy of Meth A sarcoma in mice. Tumor growth was inhibited in the adrenalectomized mice when a minimum dose of corticosterone, 0.3 mg/day, was given for replacement, and higher doses led to an even greater inhibition. Corticosterone had no effect on tumor growth in the irradiated mice. Sinecomitant immunity in the case of growth of the retransplanted excised tumor was compromised in the adrenalectomized mice. In vivo neutralization and immunosuppressive activities were absent in the spleen cells of the adrenalectomized mice. It would thus appear that adrenalectomy suppresses tumor growth by mechanisms other than glucocorticoid ablation. For optimum tumor control, glucocorticoid replacement after adrenalectomy should be in excess of the minimum daily requirements.

Biopsy tissues from a 68-year-old Japanese man with metastases to axillary lymph nodes of a recurrent esophageal carcinoma were adapted to cell culture conditions and a continuously growing tumor cell line was developed. Immunohistochemical staining revealed that these cells contained keratinous material and the electron microscopic study revealed the presence of tonofilaments. Thus, this line, designated the KSE-1 line, was considered to have originated from metastatic squamous cell carcinoma of the esophagus. This line has a binding content of 4.2 fmol/mg protein for the estrogen receptor and 2.2 fmol/mg protein for the testosterone receptor. By measurement of cell number and thymidine incorporation, the growth rate of this line was found to be moderately responsive to these hormones, being inhibited by estrogen and enhanced by testosterone at concentration levels between 10(-13) and 10(-8) and 10(-13) and 10(-6) mg/ml, respectively.

Intrapleural instillations of recombinant interleukin 2 (RIL-2) were performed in 11 patients with malignant pleurisy due to lung cancer. Kinetic studies on RIL-2 concentration in the pleural effusion and serum revealed relatively long-term maintenance of detectable levels of RIL-2 (over 24 h in the pleural effusion and over 8 h in the serum). Clinically, pleural effusions and cancer cells in the effusions disappeared in 9 of the 11 patients 4 to 10 days after the start of the treatment. Lymphokine-activated killer cells were induced in the effusions of responders who exhibited the disappearance of pleural effusion and cancer cells from the effusion, but not in those of the nonresponders. This induction of lymphokine-activated killer cells may result in the disappearance of cancer cells and pleural effusions. Cytological examination of pleural effusions revealed increases of lymphoblasts, immunoblastic large lymphocytes, and eosinophiles in number and proportion in the responder, although such a phenomenon could not be observed in the nonresponders. Main and frequent side effects of intrapleural instillations of RIL-2 were fever up to 39 degrees C, transient increase of pleural effusion, and eosinophilia. No serious side effect was encountered in our experience.

The percentage of tumor-associated macrophages recovered (TAMR) and antitumoral activity of tumor-associated macrophages (TAM) were examined in 77 patients with resectable primary lung cancer. TAM was obtained by plastic adherence following trypsinization. TAMR increased from Stage I to Stage II and decreased in Stage III. It also increased in N1 as compared with N0 and N2 but was unrelated to tumor size. However, the cytostatic activity of TAM declined with advance in stage of the disease and an increase of tumor size, but it was relatively unaffected by the presence of metastasis to regional lymph nodes. There was no correlation between TAMR and the recurrence rate; however, cytostatic activity of TAM was correlated significantly with the prognosis of totally resected cases. TAMR and cytostatic activity of TAM tended to be lower in palliatively resected cases. These results suggest that the assessment of the antitumor activity of TAM, but not merely TAMR, may give prognostic information for lung cancer patients.

Dipyridamole (DP) is clinically prescribed for its vasodilating and antiplatelet effects. DP also inhibits nucleoside transport and enhances cytostatic activity of antimetabolites. We obtained evidence for augmentation of the cytocidal effect of Adriamycin (ADM) by DP, both in vitro and in vivo. Nontoxic levels of DP enhanced the cytotoxicity of ADM against HeLa cells, and the 50% effective concentration of ADM was decreased 2.4-fold by DP. DP also increased the activity of ADM in clonogenic assays. Intracellular levels of ADM in the case of concomitant exposure to ADM and DP were 1.5-fold higher than in the case of exposure to ADM alone, determined using high-performance liquid chromatography. Incorporation of ADM into the cells pretreated with DP was also increased (1.4-fold), while the efflux was little affected. The growth of Sarcoma 180 tumors was prominently suppressed by the combination of ADM and DP, compared to findings with ADM alone. DP also prolonged the survival of Sarcoma 180 tumor-bearing mice, when given in combination with ADM. While the enhancement of cytostatic activity of antimetabolites has been attributed to a decrease in utilization of the salvage pathway by DP, our data show that the synergic effects of DP with ADM were the result of increased intracellular levels of ADM.