Clin Cancer Res

A prospective study of K-ras mutations in the plasma of pancreatic cancer patients.

Year 1998
Mulcahy HE. Lyautey J. Lederrey C. qi Chen X. Anker P. Alstead EM. Ballinger A. Farthing MJ. Stroun M.
Digestive Diseases Research Centre, St. Bartholomew's, London, United Kingdom.
K-ras mutations are frequently found in primary pancreatic adenocarcinomas. In this prospective study, we looked for K-ras mutations in the plasma of patients with pancreatic cancer. We isolated plasma DNA from 21 pancreatic cancer patients using a simple and rapid extraction technique and detected K-ras alterations with a PCR assay and subsequent product sequencing. Patients were followed up to determine their clinical outcome. We found K-ras mutations in the plasma of 17 patients (81%). In cases in which both plasma and pancreatic tissue were available, DNA mutations were similar in corresponding plasma and tissue samples. Plasma DNA alterations were found 5-14 months before clinical diagnosis in four patients. Mutant DNA was not found in the plasma of two patients with chronic pancreatitis or in five healthy controls. Our results indicate that K-ras mutations are often found in DNA isolated from the plasma of pancreatic cancer patients and that a noninvasive plasma-based assay may provide qualitative diagnostic information to clinicians in the future. Larger studies are required to further assess the relevance of our findings to clinical practice.

Quantitation of DNA from exfoliated colonocytes isolated from human stool surface as a novel noninvasive screening test for colorectal cancer.

Year 1998
Loktionov A. O'Neill IK. Silvester KR. Cummings JH. Middleton SJ. Miller R.
Dunn Clinical Nutrition Centre, Addenbrooke's Hospital, Cambridge, United Kingdom.
The only widely used screening test for early detection of colorectal cancer, the fecal occult blood test, lacks both sensitivity and specificity because it relies upon incidental bleeding rather than the neoplastic process. With the purpose of developing a new noninvasive diagnostic approach, we quantified DNA extracted from cells isolated from the surface of human stools by a novel procedure. Stools collected from 28 healthy individuals, 17 colorectal cancer patients, and 11 colorectal polyp patients were analyzed. A stool DNA index (SDNAI), expressed as DNA amount in nanograms per gram of stool, had a remarkable 4.5-fold difference in mean values between colorectal cancer patients and healthy people of comparable age. SDNAI was 2133 +/- 407 in the cancer group versus 469 +/- 65 in healthy people of the older (> 50 years) age group (P = 0.0005). The difference was independent of tumor location and size. If 700 ng of DNA/g of stool was taken as a cutoff SDNAI value in discrimination between older healthy people and cancer patients, sensitivity and specificity values reached 1.00 and 0.81, respectively. Age dependence of SDNAI was demonstrated by substantially lower SDNAI values (mean, 227 +/- 41) in younger healthy individuals. Polyp patients sometimes displayed elevated SDNAI values, but considerable variation was observed (mean, 1215 +/- 548). These preliminary findings indicate that SDNAI provides a novel, simple, and powerful noninvasive test for colorectal cancer early detection and screening. The fundamental advantage of the SDNAI is that it directly characterizes colonic epithelium involved in carcinogenesis.

Dissemination of tumor cells in patients undergoing surgery for colorectal cancer.

Year 1998
Weitz J. Kienle P. Lacroix J. Willeke F. Benner A. Lehnert T. Herfarth C. von Knebel Doeberitz M.
Division of Molecular Diagnostics and Therapy, University of Heidelberg, Germany.
The majority of patients with colorectal cancer present at a stage when the primary cancer can be resected with curative intent. However, despite the high resectability rate, about 30-50% of these patients subsequently develop metastatic disease. In these patients, neoplastic cells were disseminated either before or during surgery of the primary cancer. Due to the lack of appropriate detection systems, the extent of pre- and intraoperative hematogenic tumor cell dissemination has not yet been determined. Using a reverse transcription-PCR assay to amplify cytokeratin 20 transcripts, we were able to detect 10 colorectal cancer cells in 10 ml of blood. Blood samples were taken from 65 patients undergoing resection of primary colorectal cancer or liver metastasis of colorectal cancer pre-, intra-, and postoperatively. Circulating tumor cells were detected in 24 of 58 patients with colorectal resections in correlation to the tumor stage and in 6 of 7 patients who underwent hemihepatectomy for liver metastasis. In 8 of 58 patients with colorectal resection and in 5 of 7 patients with hemihepatectomy, tumor cells could only be detected during or during and after surgery. These results demonstrate that hematogenic tumor cell dissemination is a frequent and early event in colorectal cancer. Surgery enhances the release of tumor cells into the circulation. The long-term follow-up of our patient cohort will provide data on the prognostic relevance of circulating tumor cells and might lead to new therapeutic concepts for perioperative prophylaxis of tumor cell implantation or postoperative adjuvant therapy regimens.

Significance of platelet-derived endothelial cell growth factor in the angiogenesis of human gastric cancer.

Year 1998
Takahashi Y. Bucana CD. Akagi Y. Liu W. Cleary KR. Mai M. Ellis LM.
Department of Surgical Oncology, Kanazawa University, Japan.
We have previously shown that platelet-derived endothelial cell growth factor (PD-ECGF) is associated with angiogenesis of human colon cancer; this factor is expressed at high levels in vascular tumors that express low levels of vascular endothelial growth factor (VEGF). In these colon cancers, the major source of PD-ECGF is the infiltrating cells. In this study, we examined the role of PD-ECGF in the angiogenesis of human gastric cancer. Immunostaining for PD-ECGF was done on 93 gastric cancers previously stained for VEGF, basic fibroblast growth factor, and factor VIII-related antigen (specific for endothelial cells). To determine the cell type expressing PD-ECGF, double staining was done using antibodies to both PD-ECGF and CD68 (specific for macrophages). PD-ECGF was expressed more frequently in infiltrating cells (positive CD68 staining; 53.8%) than in tumor epithelium (9.7%; P < 0.0001). Infiltrating cells simultaneously stained positive for both PD-ECGF and CD68. An association between PD-ECGF expression in infiltrating cells, VEGF expression in tumor epithelium, and vessel count was observed in intestinal-type gastric cancer but not in diffuse-type gastric cancer. Vessel count was greater in tumors with high expression of both PD-ECGF and VEGF than in those with high expression of either factor alone (P = 0.002). Multiple angiogenic factors expressed by both tumor cells and infiltrating cells may play a role in the regulation of angiogenesis in intestinal-type gastric cancer.

Pretreatment of colon carcinoma cells with Tomudex enhances 5-fluorouracil cytotoxicity.

Year 1998
Longo GS. Izzo J. Chang YM. Tong WP. Zielinski Z. Gorlick R. Chou TC. Bertino JR.
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
The cytotoxic effect of sequence and dose of Tomudex (TX) and 5-fluorouracil (FUra) on an HCT-8 colon carcinoma cell line using a clonogenic assay was evaluated. Synergistic cell kill was obtained with 24 h of exposure to TX followed by 4 h of exposure to FUra. Marginal synergy was obtained with the same sequence but with a 5-day exposure to FUra. The reverse sequence, FUra (either 4 h or 5 days), followed by TX (24 h), resulted in less-than-additive cell kill. The synergistic effect was not due to augmented inhibition of thymidylate synthase, as determined by the measurement of thymidylate synthase activity by tritium release from [5-3H]2'-deoxyuridine. Surprisingly, an increase in intracellular levels of phosphoribosylpyrophosphate was observed after 24 h of exposure to TX, suggesting the possibility of an indirect effect of TX and/or its polyglutamates on purine biosynthesis. Moreover, we observed an increased formation of FUra nucleotides in the cells preexposed to TX, likely due to the increased intracellular levels of phosphoribosylpyrophosphate, that as a consequence led to an enhanced incorporation of FUra into RNA and increased cell killing.

CD44 expression in the stromal matrix of colorectal cancer: association with prognosis.

Year 1998
Furuta K. Zahurak M. Goodman SN. Hamilton SR. August JT.
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
CD44, a cell hyaluronate receptor, is implicated in the metastatic behavior of some cancer cells. This study analyzed CD44 expression in topographic tissue sites of colorectal cancers to determine its association with patient survival and clinicopathological characteristics. Immunohistochemical localization of the core CD44 and the v6 splice variant domains was examined by use of paraffin-fixed sections from 133 stage II or III colorectal cancers that previously had been evaluated for other diagnostic markers. Expression in malignant epithelium, stromal matrix, and stromal cells was compared to patient survival by univariate, multivariate, and bootstrap (reproducibility) analysis. Core CD44 staining was present in the malignant epithelium of 85% of tumors, the stromal matrix of 90%, and the stromal cells of 98%. The v6 splice variant domain was present in the epithelium of 77% of tumors but was less frequent in the stromal matrix (12%; P < 0.001) and stromal cells (17%; P < 0.001). Absence of core CD44 immunoreactivity in the stromal matrix was associated with increased death rate (hazard ratio, 2.4; 95% confidence interval, 1.2-4.8; P = 0.02), making this one of the most significant adverse prognostic variables, along with an age of 60 years or older, poor differentiation of the cancer, extramural venous invasion, chromosome 18q allelic loss, and nonwhite race. This study shows that core CD44 and v6 splice variant antigens are differentially expressed in the epithelium and stroma of colorectal cancers. A model that includes core CD44 immunoreactivity in stromal matrix along with other prognostic factors may improve identification of high-risk and low-risk patients.

TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival.

Year 1998
Borresen-Dale AL. Lothe RA. Meling GI. Hainaut P. Rognum TO. Skovlund E.
Department of Genetics, Norwegian Radium Hospital, Oslo, Norway. alb@radium.uio.no
In a consecutive series of 222 colorectal carcinomas from patients with a median follow-up time of 56.8 months (range, 0.5-92.2) treated with surgery, the TP53 gene was screened for mutations. Exons 5-8 were analyzed using constant denaturant gel electrophoresis followed by sequencing, and mutations were found in 102 cases (45.9%). Mutations were found more frequently in rectal tumors versus other locations (P = 0.029) and in aneuploid compared to diploid tumors (P < 0.001). Presence of a TP53 mutation was also significantly associated with absence of microsatellite instability (P = 0.028), as well as with loss of heterozygosity at 17p13 (P < 0.001). The TP53 mutations in the left-sided and rectal tumors were more often transversions than transitions, indicating a different etiology in the development of these tumors. The tendency for shorter cancer-related survival among patients with mutations in their tumors was only statistically significant for patients with left-sided tumors (P = 0.003). All patients with mutations affecting the L3 domain of the protein involved in zinc binding had a significantly shorter cancer-related survival (P = 0.036), indicating that mutations affecting this domain have biological relevance in terms of colorectal cancer disease course. These results suggest that knowledge of a patient's TP53 status, with respect to both the presence and the localization of the mutation, may be important in prognosis evaluation, particularly when selecting patients for more aggressive postoperative therapeutic intervention.

Pharmacodynamics of topoisomerase I inhibition: Western blot determination of topoisomerase I and cleavable complex in patients with upper gastrointestinal malignancies treated with topotecan.

Year 1998
Liebes L. Potmesil M. Kim T. Pease D. Buckley M. Fry D. Cho J. Adler H. Dar K. Zeleniuch-Jacquotte A. Hochster H.
Kaplan Cancer Center, New York University Medical Center, New York 10016, USA.
Analogues of camptothecins are specific inhibitors of eukaryotic DNA topoisomerase I (topo I) that lead to DNA damage and, eventually, cellular cytotoxicity. Camptothecin analogues bind to this target enzyme in the course of its normal function and stabilize the DNA-enzyme adduct to form a "cleavable complex." Preclinical experiments using Western blot analyses have shown cleavable complex formation to be the key intermediate step in topo I inhibition. In this series of experiments, it was our goal to convert this laboratory technique into a useful clinical assay, allowing measurement of the target enzyme and detection of the key intermediate in clinical specimens taken from patients being treated with the topo I inhibitor topotecan. Because available antibodies were not sufficiently sensitive at the start of this project, we identified a highly specific human SCL-70 antibody from a patient with scleroderma, which allowed quantitative determination of topo I copy number in HeLa and HT-29 cell lines. Additional refinements of the Western blot technique were accomplished to improve signal:noise ratio. In surgical tumor specimens, we found the median topo I level to be 30.1 x 10(5) copies/cell for gastric adenocarcinomas, compared to 18.4 x 10(5) copies/cell for normal gastric mucosae in the same samples. For lung adenocarcinoma, the median protein level was 21.5 x 10(5) copies/cell, compared with the normal tissue counterpart protein level of 12.7 x 10(5) copies/cell. The median tumor:normal ratios from paired samples of these tumor types were 1.51 and 1.84, respectively. As part of a Phase II study evaluating the efficacy of topotecan (1.5-2.0 mg/m2 daily for 5 days) in upper gastrointestinal malignancies, we obtained tumor and normal mucosa biopsies in 11 patients with gastric or esophageal cancer, 30 min after administration on day 4 or 5. Three patients with gastric adenocarcinoma had stable disease as their best response, with the remainder of patients progressing. Improvement in Western blotting methodology allowed the quantitation of topo I levels in these gastric and esophageal cancer biopsies, which could be augmented by brief heating to release complexed topo I. We were also able to directly visualize high molecular weight topo I-containing bands, which were shown to be cleavable complexes by heat reversal, with restoration of the topo I Mr 100,000 band. Using this heat reversal technique, we determined the presence of cleavable complex in a total of 7 of 11 patient biopsy samples (5 tumors and 2 normal mucosae). In patients treated with topotecan on this dose and schedule, we determined that a median of 73% of the total tumor topo I was involved in cleavable complex (range, 18.3-91%). The intensity of the Mr 100,000 topo I band in biopsy specimens of patients receiving topotecan represented "free" or noncomplexed topo I. The median copy number for the residual, noncomplexed topo I (n = 11) was 7.36 x 10(5) copies/cell, significantly less than the median of 30.1 x 10(5) copies/cell for random tumor specimens from patients with gastric adenocarcinomas (P < 0.001). Pharmacodynamic analysis demonstrated a negative correlation between the noncomplexed topo I copy number and topotecan area under the curve (Spearman rank test: r(s) = -0.81, P = 0.003). Nonlinear regression analyses of these data were best fit with an inhibitory maximum effect model, yielding parameter estimates for Emax and EC50 of 29.3 x 10(5) copies/cell (coefficient of variation = 22%) and 43.1 ng x h/ml (coefficient of variation = 27%), respectively. Through a series of careful modifications and refinements, we have improved the Western blot assay for topo I for use in clinical monitoring. We have demonstrated the ability to directly visualize cleavable complex in patients being treated with topo I inhibitor therapy and have directly quantitated free topo I, as well as the key topo I intermediate (cleavable complex), in biopsy specimens obtained from pat

Expression of Bcl-X(L), an antiapoptotic member of the Bcl-2 family, in esophageal squamous cell carcinoma.

Year 1998
Torzewski M. Sarbia M. Heep H. Dutkowski P. Willers R. Gabbert HE.
Department of Pathology, Heinrich-Heine University, Dusseldorf, Germany.
Bcl-X, a Bcl-2-related protein, is a potent antagonist of apoptosis in its long splice variant (Bcl-X(L)). The present study was performed to determine its expression in preneoplastic and neoplastic lesions of the esophagus, its correlation with other members of the Bcl-2 family, and its impact on the outcome of surgically treated esophageal cancer patients. Samples of normal esophageal squamous epithelium (n = 10), severe squamous cell dysplasias (n = 19), carcinomas in situ (n = 14), invasive squamous cell carcinomas (n = 172), and lymph node metastases (n = 21) were immunohistochemically analyzed for Bcl-X(L) expression using a polyclonal anti-Bcl-X(L) antibody. The immunostaining was evaluated according to a score system (0-12 points) based on the percentage of positive tumor cells and the relative immunostaining intensity. Cytoplasmic staining for Bcl-X(L) protein was invariably found in all cell layers of the normal esophageal squamous epithelium. In contrast, a considerable portion of preneoplastic and neoplastic lesions display a decreased Bcl-X(L) expression as compared with that in the normal esophageal epithelium. On comparison of the amount of Bcl-X(L) expression between the different types of lesions, however, no significant differences were found between severe squamous cell dysplasias (mean immunoreactive score +/- SD, 5.2 +/- 1.8), carcinomas in situ (5.2 +/- 2.2), invasive carcinomas (4.5 +/- 2.8), and lymph node metastases (4.2 +/- 2.6). In invasive carcinomas, Bcl-X(L) expression decreased continuously with decreasing tumor differentiation (P = 0.0001) and was also directly correlated with bcl-2-associated X protein expression (P = 0.0001). On the contrary, an inverse correlation was found between Bcl-X(L) expression and Bcl-2 protein expression (P = 0.0001). No correlation was found between Bcl-X(L) expression and the parameters pT category, pN category, and tumor size. In the univariate survival analysis, patients with low immunoreactive scores (< or = 4) of Bcl-X(L) expression in the tumor tissue showed lower 2-year and 5-year survival rates than patients with high immunoreactive scores (> 4; P = 0.0485). In multivariate survival analysis, however, only the parameters pN category and pT category, but not Bcl-X(L) expression, could be verified as independent prognostic factors. This tendency of decreasing levels of an antiapoptotic protein toward unfavorable outcome is supported by an increasing number of studies on the role of Bcl-2, another antiapoptotic protein, and must be interpreted against the backdrop of apoptosis as a result of the interaction of many cell death-promoting and protecting proteins.

Bispecific antibodies increase T-cell stimulatory capacity in vitro of human autologous virus-modified tumor vaccine.

Year 1998
Haas C. Strauss G. Moldenhauer G. Iorio RM. Schirrmacher V.
Division of Cellular Immunology, German Cancer Research Center, Heidelberg.
The production and functional testing of two new bispecific (bs) hybrid antibodies [Abs; bs Ab hemagglutinin-neuraminidase (HN) x CD3 and bs Ab HN x CD28] designed for cancer vaccine modification are described. They allow distinct modifications of the human tumor cell vaccine ATV-NDV, an autologous tumor cell vaccine already modified by infection with Newcastle disease virus. The bs Abs use the viral HN molecule as a common foreign anchoring molecule for attachment to the tumor cells and allow the introduction of anti-CD3 or anti-CD28 T-cell-stimulatory molecules. The bs Abs attached to tumor target cells were able to cross-link CTL effector cells and up-regulate T-cell activation markers on autologous cancer patient-derived CD4 and CD8 T lymphocytes. This strategy of combining a cellular vaccine with a bs Ab is highly specific, quick, and economical and has broad-range applications. Five ng or less of target cell-bound bs Ab HN x CD28 were effective at augmenting T-cell-mediated antitumor cytotoxicity.

Mechanisms for ganciclovir resistance in gastrointestinal tumor cells transduced with a retroviral vector containing the herpes simplex virus thymidine kinase gene.

Year 1998
Yang L. Hwang R. Chiang Y. Gordon EM. Anderson WF. Parekh D.
Gene Therapy Laboratories, Los Angeles, California, USA.
Transfer of the herpes simplex thymidine kinase (HSV-TK) gene into tumor cells confers sensitivity to the cells to the viral drug ganciclovir (GCV). Although the efficacy of the HSV-TK/GCV approach is well studied, the mechanisms for the resistance of HSV-TK-transduced tumor cells to GCV are poorly understood. Here, we examined the mechanisms for GCV resistance in HSV-TK-transduced gastrointestinal (GI) cell lines. Our results show that GCV sensitivities vary in vitro and in vivo among the different HSV-TK-transduced GI tumor cell lines. GCV-resistant colonies were isolated from several different HSV-TK-transduced GI tumor cell lines after 14 days of GCV treatment. Characterization of GCV-resistant colonies demonstrated that the HSV-TK gene was either partially or completely deleted from the resistant HSV-TK-transduced cells. In the HT-29 RM and MIAPACA-2 RM cells, a 220-bp deletion of the gene was found, whereas in the HT-29 R1-R5-resistant cells, the whole TK gene was found to be absent. Immunocytochemical studies using a polyclonal antibody to the TK protein demonstrated that the HSV-TK protein was absent in the GCV-resistant, HSV-TK-transduced cells. Transfection of the resistant cells with an adenoviral vector containing a HSV-TK gene restored sensitivity to GCV. The presence of GCV-resistant cells was only demonstrable in GI tumor cell lines that also demonstrated a poor bystander effect. Our results suggest that GCV resistance found in tumor cells transduced with a retroviral HSV-TK gene is due to the lack of a functional TK protein in the tumor cells rather than any intrinsic resistance of the cells to GCV. In tumor cells with a good bystander effect, the small percentage of TK-transduced cells that do not express the TK protein are probably killed by the bystander effect because GCV-resistant tumor cells were not found in these cell lines. GCV-resistant tumor cells were found only in tumor cell lines with a poor bystander effect, by which, presumably, the transduced tumor cells lacking a functional TK gene were not killed by the bystander killing effect.

Studies of the efficacy and pharmacology of irinotecan against human colon tumor xenograft models.

Year 1998
Zamboni WC. Stewart CF. Cheshire PJ. Richmond LB. Hanna SK. Luo X. Poquette C. McGovren JP. Houghton JA. Houghton PJ.
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Irinotecan, administered i.v. on days 1-5 and 8-12 [(dx5)2 i.v.] has demonstrated significant activity against advanced human tumor xenografts. To explore the feasibility of prolonged oral administration of irinotecan, we compared the efficacy of oral and i.v. irinotecan on the (dx5)2 schedule. We also evaluated oral therapy for 12 consecutive weeks [(dx5)12] at 25 and 50 mg/kg and two consecutive 5-day courses repeated every 21 days for up to four cycles ([(dx5)2]4) at 50 and 75 mg/kg/dose in a series of human colon carcinoma xenograft lines. In addition, we evaluated the effect of a sensitive (HC1) and resistant (ELC2) human colon adenocarcinoma xenograft on irinotecan and SN-38 lactone disposition after administration of irinotecan 10 mg/kg i.v. and 10 and 25 mg/kg p.o. Irinotecan i.v. at 40 mg/kg and oral at 50 and 75 mg/kg on the (dx5)2 schedule had similar activity against the panel of adult colon adenocarcinoma xenografts. Irinotecan given p.o. also demonstrated significant activity against a topotecan-resistant derivative, VRC5/TOPO. Oral administration of 75 mg/kg [(dx5)2]4 and 50 mg/kg (dx5)12 achieved complete response in five of seven xenograft lines evaluated. After i.v. administration, mice bearing HC1 xenografts had 43% greater SN-38 lactone systemic exposure compared to those with ELC2 xenografts and non-tumor-bearing mice. After oral (10 mg/kg) administration, there was a 5-fold higher molar formation of SN-38 lactone compared to i.v. (10 mg/kg) administration in tumor and non-tumor-bearing mice. SN-38 systemic exposure associated with the lowest oral dose (25 mg/kg) achieving complete response for HC1 was 942.6 ng/ml x h. These results emphasize the importance of pharmacokinetic studies as part of tumor response studies in xenograft models.

Lineage-negative human leukocyte antigen-DR+ cells with the phenotype of undifferentiated dendritic cells in patients with carcinoma of the abdomen and pelvis.

Year 1998
Melichar B. Savary C. Kudelka AP. Verschraegen C. Kavanagh JJ. Edwards CL. Platsoucas CD. Freedman RS.
Department of Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
The characteristics of antigen-presenting cells in carcinomas that involve the abdominopelvic cavity are unknown. Dendritic cells, a population of antigen-presenting cells, have been identified as lineage-negative human leukocyte antigen (HLA)-DR+ cells by two-color flow cytometry. We used this criterion to study the putative dendritic cells in ascites from 25 patients with peritoneal carcinomatosis. The mean proportion +/- SD of lineage-negative HLA-DR+ cells in ascites was 3.1 +/- 4.6% (range, 0.05-17.3%). Most lineage-negative HLA-DR+ cells expressed CD45RA or CD4 antigens. Dendritic cells had low proportions of CD80, CD11c, CD45RO, and CD58, suggesting that they were of low maturity. The proportion of lineage-negative HLA-DR+ cells in ascites of seven patients was significantly higher than the proportion in peripheral blood from the identical patients (4.5 +/- 5.7 versus 0.5 +/- 0.4; P < 0.05). In paired specimens of ascites and peripheral blood, the proportion of lineage-negative HLA-DR+ cells that coexpressed CD86 or CD58 was significantly lower in ascites than in peripheral blood, whereas a higher proportion of lineage-negative HLA-DR+ cells in ascites expressed CD4. Relative fluorescence intensity of HLA-DR+ was also lower in dendritic cells from ascites and blood from patients with carcinomatosis than it was in blood from normal donors. As an indicator of macrophage activation, the concentration of neopterin in ascitic fluid correlated negatively with the numbers of lineage-negative HLA-DR+ cells in ascites (Spearman correlation coefficient, -0.44; P = 0.05) correlated positively with the concentration of interleukin 10 in ascitic fluid (Spearman correlation coefficient, -0.40; P = 0.05). IFN-gamma and tumor necrosis factor alpha were also not detected. These findings suggest that certain factors associated with the tumor microenvironment might influence the number of these dendritic cells and their expression of function-associated markers.

Quantitative analysis of carcinoembryonic antigen messenger RNA in peripheral venous blood and portal blood of patients with pancreatic ductal adenocarcinoma.

Year 1998
Funaki NO. Tanaka J. Hosotani R. Kogire M. Suwa H. Imamura M.
Department of Surgery, Shiga Medical Center for Adults, Moriyama City, Japan.
One major therapeutic failure of pancreatic adenocarcinoma treatment is metastasis to the liver. To screen patients with high risk for such hematogenous dissemination, we previously developed a very sensitive system to detect carcinoembryonic antigen (CEA) in blood. For a more practical application, we improved this system by making it quantitative and capable of analyzing both preoperative peripheral blood and intraoperative portal blood for the presence of CEA mRNA. CEA mRNA was not detected in the peripheral venous blood of any of the three patients examined, but it was identified in the portal blood without fail. In addition, the quantities of CEA mRNA identified in the portal blood before and after pancreatectomy were different. This study suggests that analysis of the portal blood seems to be important for the precise evaluation of hematogenous dissemination and of the pathophysiology of pancreatic ductal adenocarcinoma.

Analysis of colorectal cancer by comparative genomic hybridization: evidence for induction of the metastatic phenotype by loss of tumor suppressor genes.

Year 1998
Paredes-Zaglul A. Kang JJ. Essig YP. Mao W. Irby R. Wloch M. Yeatman TJ.
Department of Surgery, H. Lee Moffitt Cancer Center, University of South Florida, Tampa 33612, USA.
Current models suggest that colon cancer initiation and progression are secondary to both the activation of oncogenes and the deletion of tumor suppressor genes. The role of each, however, is still poorly understood, particularly with regard to the induction of metastasis. We hypothesized that genetic differences exist between tumors that metastasize distantly and those that do not, and that oncogenes and tumor suppressor genes participate equally in this process. To address this hypothesis, human tumor specimens from localized [tumor-node-metastasis (TNM) stage I-III] and primary colon cancers (n = 10) were directly compared with metastatic (TNM stage IV) lesions (n = 10) using comparative genomic hybridization analysis. Although several alterations were shared equally between primary tumors and metastases (+7q, +19q, and +20q), two patterns of distinguishing alterations were observed: (a) alterations that were more extensive in liver metastases than in primary tumors (+8q, +13q, -4p, -8p, -15q, -17p, -18q, -21q, and -22q); and (b) alterations that were unique to metastatic lesions (-9q, -11q, and -17q). Overall, genetic losses were more common than gains, and, more importantly, the number of losses/tumor was significantly higher for metastases than for primary tumors (9.3 + 1.3 versus 4.1 + 0.7; P = 0.00062, Wilcoxon's rank-sum test). The distinct predominance of genetic losses in the metastatic lesions when compared with the primary localized tumors provides evidence that the metastatic phenotype is induced by the deletion of tumor suppressor genes and permits the construction of physical maps targeting regions where novel tumor suppressor genes are likely to exist.

Phase II and pharmacodynamic studies of pyrazine diazohydroxide (NSC 361456) in patients with advanced renal and colorectal cancer.

Year 1998
Vogelzang NJ. Mani S. Schilsky RL. Ansari RH. Taber D. Rhinehart SN. Garcia JC. Meyer SC. Mick R. Brockstein BE. Stadler WM. Ratain MJ. Vokes EE.
Cancer Research Center, University of Chicago Pritzker School of Medicine, Illinois 60637-1470, USA. njvogelz@mcis.bsd.uchicago.edu
Pyrazine diazohydroxide (PZDH) is a novel antitumor agent that forms DNA adducts via the reactive pyrazine diazonium ion. In a recent Phase I study of PZDH, we identified a recommended Phase II dose of 100 mg/m2/day x 5, given as a 5-min i.v. bolus with the cycles repeated every 42 days (N. J. Vogelzang, et al, Cancer Res., 54: 114-119, 1994). There was a moderate negative correlation between serum chloride concentration and logarithm platelet nadir, suggesting the hypothesis that PZDH is activated in an acidic environment, leading to more toxicity in acidotic patients. Therefore, the University of Chicago Phase II cooperative network conducted two Phase II studies of PZDH in renal cancer (15 patients, 2 with liver metastases) and in 5-fluorouracil-refractory colorectal cancer (14 patients, 13 with liver metastases) to determine efficacy in each disease and to correlate safety and tolerance of the drug with PZDH pharmacokinetics/pharmacodynamics and with arterial blood gas measurements. There were no responses seen in either tumor type. The primary toxicity of PZDH was myelosuppression with neutropenia (absolute neutrophil count, < 1000/microl) and thrombocytopenia (

Prospective randomized trial of 5-fluorouracil versus 5-fluorouracil plus levamisole in the treatment of metastatic colorectal cancer: a Hoosier Oncology Group trial.

Year 1998
Bandealy MT. Gonin R. Loehrer PJ. Monaco F. Einhorn LH.
Division of Hematology/Oncology, Indiana University, Indianapolis 46208-4646, USA.
The purpose of this study was to compare the objective response rate, duration of remission, and survival of 5-fluorouracil (5-FU) versus those of 5-FU plus levamisole in metastatic colorectal cancer using the same dose and schedule of these agents as in the North Central Cancer Treatment Group and intergroup studies of adjuvant therapy. Patients with no prior history of chemotherapy for metastatic disease were entered on this Hoosier Oncology Group randomized Phase III trial. Patients were stratified by Karnofsky performance status and presence or absence of liver metastases. They were randomized to receive 450 mg/m2 5-FU i.v. for 5 days followed by 15 mg/kg i.v. weekly (arm 1) or the same dose of 5-FU plus levamisole 50 mg p.o. every 8 h for 3 days every 2 weeks (arm 2). The duration of treatment for both arms was 26 weeks. From April 1990 to March 1995, 199 patients were entered. One hundred eighty-two patients, 91 in each arm, were fully evaluable. The response rates were 12% on arm 1 and 13% on arm 2. The median duration of response was 18 weeks on both arms. The median survival was 48 weeks on arm 1 and 41 weeks on arm 2 (P = 0.20). This study failed to show any improvement in survival, response, or duration of remission with the addition of levamisole to 5-FU in the treatment of metastatic colorectal cancer.

Effect of food on the pharmacokinetics of capecitabine and its metabolites following oral administration in cancer patients.

Year 1998
Reigner B. Verweij J. Dirix L. Cassidy J. Twelves C. Allman D. Weidekamm E. Roos B. Banken L. Utoh M. Osterwalder B.
Department of Clinical Pharmacology, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Capecitabine (Ro 09-1978) is a novel oral fluoropyrimidine carbamate that was rationally designed to generate 5-fluorouracil (5-FU) selectively in tumors. The effect of food on the pharmacokinetics of capecitabine and its metabolites was investigated in 11 patients with advanced colorectal cancer using a two-way cross-over design with randomized sequence. Patients received repeated doses of 666 or 1255 mg/m2 of capecitabine twice daily. On study days 1 and 8, drug was administered following an overnight fast or within 30 min after consumption of a standard breakfast, and serial blood samples were collected. Concentrations of capecitabine and its metabolites [5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), 5-FU, dihydro-5-fluorouracil (FUH2), and alpha-fluoro-beta-alanine (FBAL)] in plasma were determined by high-performance liquid chromatography or liquid chromatography/mass spectroscopy. Intake of food prior to the administration of capecitabine resulted in pharmacokinetic changes of all compounds involved. The extent of these changes, however, varied considerably between the various compounds. Maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) values were decreased after food, and time until the occurrence of Cmax values were increased. In contrast, the apparent elimination half-life was not affected by food intake. The extent of change in Cmax and AUC was highest for capecitabine and decreased with the order of formation of the metabolites. The "before:after food" ratios of the Cmax values were 2.47 for capecitabine, 1.81 for 5'-DFCR, 1.53 for 5'-DFUR, 1.58 for 5-FU, 1.26 for FUH2, and 1.11 for FBAL. The before: after food ratios of the AUC values were 1.51 for capecitabine, 1.26 for 5'-DFCR, 1.15 for 5'-DFUR, 1.13 for 5-FU, 1.07 for FUH2, and 1.04 for FBAL. The results show that food has a profound effect on the AUC of capecitabine, a moderate effect on the AUC of 5'-DFCR, and only a minor influence on the AUC of the other metabolites in plasma. In addition, a profound influence on Cmax of capecitabine and most of its metabolites was found. Detailed information on the relationship between concentration and safety/efficacy is necessary to evaluate the clinical significance of these pharmacokinetic findings. At present, it is recommended that capecitabine be administered with food as this procedure was used in the clinical trials.

Loss of heterozygosity on chromosome 18q in cohesive-type gastric cancer is associated with tumor progression and poor prognosis.

Year 1998
Inoue T. Uchino S. Shiraishi N. Adachi Y. Kitano S.
Department of Surgery I, Oita Medical University, Hasamamachi, Japan.
Although loss of heterozygosity (LOH) on chromosome 18q is frequently found in gastric cancer, the clinical significance of this abnormality has not been well documented. We examined LOH on chromosome 18q22-23 in DNA extracted from the tissues of gastric cancer patients using the PCR-based dinucleotide repeat assay with two microsatellite markers, D18S61 and D18S58. We investigated LOH in 100 samples of DNA extracted from formalin-fixed, paraffin-embedded tissues of cohesive-type gastric cancer patients operated on between 1984 and 1993. Thirty-two of 83 informative cases (39%) showed LOH on chromosome 18q22-23 at one or two loci. The LOH correlated significantly with serosal invasion of the tumor (P = 0.004) and hematogenous recurrence (P = 0.035). In 60 cases who were cured, the 5-year survival rate in patients with LOH (54%) was lower than that in patients without LOH (81%; P = 0.019). These results suggest that 18q22-23 LOH in cohesive gastric cancer is associated with tumor progression and a patient's poor prognosis.

Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo.

Year 1998
Yang D. Kuan CT. Payne J. Kihara A. Murray A. Wang LM. Alimandi M. Pierce JH. Pastan I. Lippman ME.
Lombardi Cancer Center, Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.

Induction of thymidine phosphorylase activity and enhancement of capecitabine efficacy by taxol/taxotere in human cancer xenografts.

Year 1998
Sawada N. Ishikawa T. Fukase Y. Nishida M. Yoshikubo T. Ishitsuka H.
Cytostatics Group, Nippon Roche Research Center, Kamakura-city, Kanagawa, Japan.
Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the cytostatics capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) and its intermediate metabolite [5'-deoxy-5-fluorouridine (5'-dFUrd)] to 5-fluorouracil in tumors. We have tried to identify the best partners of capecitabine in combination therapy, such as dThdPase up-regulators, which may enhance the efficacy of this compound. Among various cytostatics studied with the WiDr human colon cancer xenograft model, Taxol, Taxotere, and mitomycin C greatly increased levels of human dThdPase in tumors, and cyclophosphamide slightly increased the enzyme level. These cytostatics simultaneously increased the levels of human tumor necrosis factor alpha (TNFalpha), which is an up-regulator of dThdPase. In cultures of the WiDr cells, however, Taxol did not up-regulate TNFalpha to a detectable level and only slightly enhanced levels of dThdPase. These results suggest that Taxol might indirectly elevate TNFalpha in tumor cells, which in turn up-regulated dThdPase in the tumor cells in the WiDr cancer xenograft. In the combination therapy, the efficacy of Taxol and Taxotere with either capecitabine or 5'-dFUrd was more than just additive. In contrast, Taxol and either 5-fluorouracil or UFT (a mixture of tegafur and uracil) in combination showed only additive activity. Taxol and Taxotere might enhance the efficacy of capecitabine and 5'-dFUrd, probably by modulating dThdPase activity in tumor tissues.

Disruption of p53 function in immortalized human cells does not affect survival or apoptosis after taxol or vincristine treatment.

Year 1998
Fan S. Cherney B. Reinhold W. Rucker K. O'Connor PM.
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
In the present study, we report our findings on the impact of p53 disruption on the sensitivity of human cell lines to the antimitotic agents Taxol and vincristine. Comparisons of cell survival and apoptosis were made with y-irradiation and, in some cases, several other DNA-damaging chemotherapeutic agents. Studies in eight Burkitt's lymphoma and lymphoblastoid cell lines (four wild-type p53 and four mutant p53 cell lines) revealed that the DNA-damaging agents assayed tended to exhibit less growth inhibition in the mutant p53 cell lines compared to the wild-type p53 cell lines. In contrast, no significant correlation was apparent between p53 gene status and the growth-inhibitory potency of Taxol or vincristine in these eight cell lines. We also found that contrary to gamma-irradiation, Taxol and vincristine could induce apoptosis in lymphoma cell lines harboring p53 mutations. These observations were explored further in lymphoblastoid VDSO cells (wild-type p53) from a normal individual and stably transfected VDSO derivatives lacking p53 function due to expression of the human papillomavirus type-16 E6 gene. We found that p53 disruption in VDSO/E6 cells blocked y-ray-induced apoptosis and afforded a survival advantage to VDSO/E6 cells compared to control-transfected cells. In contrast, p53 disruption did not affect Taxol- or vincristine-induced apoptosis or survival in VDSO cells. The effect of p53 disruption on Taxol sensitivity was explored further in the breast carcinoma MCF-7 and colon carcinoma HCT-116 cell lines that had been stably transfected with either the human papillomavirus type-16 E6 gene or a dominant-negative mutant p53 gene. Again, in these cell model systems, we found that p53 disruption did not affect the growth-inhibitory potency of Taxol. Taken together, our results suggest that p53 status is not a dominant factor in the mechanism by which antimitotic agents induce apoptosis and reduce survival in immortalized human cell lines.

The BAX gene, the promoter of apoptosis, is mutated in genetically unstable cancers of the colorectum, stomach, and endometrium.

Year 1998
Ouyang H. Furukawa T. Abe T. Kato Y. Horii A.
Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan.
Disruption of the DNA mismatch repair system, characterized by microsatellite instability (MI), plays an important role in the course of human carcinogenesis by increasing the rate of mutations of genes associated with cancers. However, it is not clear which genes are the target genes for mutation in the course of carcinogenesis. Microsatellites within the coding region of the transforming growth factor beta receptor type II (RII) and insulin-like growth factor II receptor (IGF-IIR) genes were reported to be targets for mutation during the course of carcinogenesis in MI+ tumors. Recently, somatic mutations were found in a poly(G)8 tract in the BCL-2-associated X protein (BAX) gene, one of the essential players in apoptosis, in some MI+ tumors. We examined mutations of BAX in MI+ cancers of various organs and found frameshift mutations at the poly(G)8 tract in 5 of 15 (33%) gastric cancers, 3 of 26 (12%) endometrial cancers, and 9 of 22 (41 %) colorectal cancers. In contrast, no such mutations were found in pancreatic cancer. These results suggest that mutations of BAX play an important role in the course of carcinogenesis in the stomach, colorectum, and endometrium.

Infrequent expression of p21 is related to altered p53 protein in pancreatic carcinoma.

Year 1998
Hu YX. Watanabe H. Ohtsubo K. Yamaguchi Y. Ha A. Motoo Y. Okai T. Sawabu N.
Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, Yoneizumi, Japan.
This study is designed to investigate the expression of p21 and its relation to altered p53 protein in pancreatic carcinoma (PC). Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections of PC was performed using a monoclonal antibody against p21 (187), with a parallel examination of altered p53 protein. The expression of p21 was only found in 12 of 58 (20.7%) PCs and, moreover, was mainly restricted to the well-differentiated ductal epithelium. Sixty-four % (37 of 58) of PCs showed positive p53 staining, and this change was significantly related to the absence of p21 expression (P < 0.01). In a subgroup, the proportion of the undetectable p21 expression and the expression of p53 were increased with increasing tumor grade but decreased with advancing clinical stage. The results of the present study suggest that the absence of p21 expression is very common in PCs and appears to relate to altered p53 protein. Moreover, the abnormalities involving the expression of p21 and p53 may play a more important role in the development than in the progression of this malignancy.

Evaluation of the balance between angiogenic and antiangiogenic circulating factors in patients with breast and gastrointestinal cancers.

Year 1998
Morelli D. Lazzerini D. Cazzaniga S. Squicciarini P. Bignami P. Maier JA. Sfondrini L. Menard S. Colnaghi MI. Balsari A.
Division of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy.
Angiogenesis is a critical determinant of tumor growth. Tumor cells produce or induce angiogenic molecules that act specifically on endothelial cells (ECs) but also release angiostatic molecules. Thus, tumor angiogenesis represents a net balance between positive and negative regulators of neovascularization. Sera from patients with breast or gastrointestinal cancers were evaluated for their capacity to selectively modulate the proliferation of human umbilical vein ECs; sera from 15 of 78 (19%) breast cancer patients and 8 of 53 (15%) gastrointestinal cancer patients induced human umbilical vein EC growth, whereas sera from 4 of 78 (5%) breast cancer patients and 1 of 53 (2%) gastrointestinal cancer patients inhibited EC proliferation. Growth-stimulatory sera were significantly more frequent among postmenopausal (14 of 53) than premenopausal (1 of 25) breast cancer patients; inhibitory activity was observed in 3 of 25 premenopausal patients versus 1 of 53 postmenopausal individuals. The half-life of serum-stimulating and -inhibiting factors seemed to differ, because stimulatory activity but not inhibitory activity was decreased at 5 days after surgery. The levels of vascular endothelial growth factor were elevated in about 45% of patients with growth-stimulatory sera, whereas the serum inhibition of EC growth was found to be due, at least in part, to high levels of soluble thrombospondin.

p53 and thymidylate synthase expression in untreated stage II colon cancer: associations with recurrence, survival, and site.

Year 1998
Lenz HJ. Danenberg KD. Leichman CG. Florentine B. Johnston PG. Groshen S. Zhou L. Xiong YP. Danenberg PV. Leichman LP.
Division of Medical Oncology, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA. lenz@hsc.usc.edu
We initiated a retrospective study to determine whether p53 status and thymidylate synthase (TS) protein expression in primary colon tumors influence recurrence and survival for patients with stage II colon cancer. Tumor specimens from 45 consecutive untreated patients with stage II colon cancer were examined for p53 and TS protein expression using immunohistochemistry. The median follow-up was 5.1 years. Eighteen patients had left-sided tumors, and 27 had right-sided tumors. Fourteen of 45 patients (31%) developed recurrence. p53 overexpression was detected in the tumors of 18 patients (40%); 10 patients (55%) with p53 overexpression recurred; and 4 of 27 (15%) without evidence of p53 overexpression recurred (P = 0.002). High TS expression was detected in the tumors of 16 patients (36%): 8 patients (50%) with high TS expression recurred, and 6 patients (21%) with low TS expression recurred (P = 0.027). Patients with p53 overexpression had a significantly poorer survival than did those patients without p53 overexpression (P < 0.001). High TS expression was associated with poor survival (P = 0.004). p53 overexpression and high TS expression were significantly associated with left-sided tumors (P = 0.003 and P = 0.022). Thirteen of 16 patients (81%) with high TS expression also overexpressed p53, and 24 of 29 patients (81%) with low TS expression did not manifest p53 overexpression (P < 0.001). p53 and TS expression in primary stage II colon cancer are associated and appear to influence recurrence and survival. In this pilot study, left-sided tumors demonstrate significantly more p53 overexpression and significantly higher TS expression than do right-sided tumors, which may explain the significantly poorer survival for patients with left-sided tumors.

p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival.

Year 1998
Lenz HJ. Hayashi K. Salonga D. Danenberg KD. Danenberg PV. Metzger R. Banerjee D. Bertino JR. Groshen S. Leichman LP. Leichman CG.
University of Southern California/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA. lenz@hsc.usc.edu
Recent studies suggest that there may be a strong correlation between the p53 status of a tumor and a patient's response to chemotherapy. Therefore, we determined p53 status in 36 patients with disseminated colorectal cancer by cDNA sequencing and immunohistochemical staining, as well as by the gene expression level of thymidylate synthase (TS), the target enzyme of 5-fluorouracil (5-FU), by reverse transcription-PCR. Ten patients (28%) experienced a clinical response to 5-FU chemotherapy. Overall, TS expression and response to chemotherapy were associated: 9 of 18 (50%) patients with TS < or = 3.0 x 10(-3) responded, compared to 1 of 18 (6%) patients with TS > 3.0 x 10(-3) (P = 0.003). p53 mutations were found in 21 of 36 patients (58%) using cDNA cycle sequencing, and p53 protein overexpression was found in 20 of 32 patients (62%) using immunohistochemistry staining. Overall p53 status and response to chemotherapy were associated: 5 of 10 (50%) patients with wild-type p53 or negative p53 staining experienced a response, but only 5 of 26 (19%) patients with mutant p53 or p53 overexpression responded. TS expression, but not expression of p53, was significantly associated with overall survival (P = 0.002). Patients with wild-type p53 had significantly lower TS levels compared to patients with mutated p53 (P = 0.044). In this study, we also present data linking specific p53 point mutations to TS expression levels and resistance to 5-FU. Although the number of patients is relatively small, these results identify p53 status and TS gene expression as associated with response in disseminated colorectal cancer; independent studies are needed to confirm these findings and to provide information leading to a better understanding of the role of 5-FU-based chemotherapy in the treatment of colorectal cancer.

Loss of p21WAF1/Cip1 protein expression accompanies progression of sporadic colorectal neoplasms but not hereditary nonpolyposis colorectal cancers.

Year 1998
Sinicrope FA. Roddey G. Lemoine M. Ruan S. Stephens LC. Frazier ML. Shen Y. Zhang W.
Department of Gastrointestinal Medical Oncology & Digestive Diseases, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.
p21 (p21WAF1/Cip1), a cyclin-dependent kinase inhibitor, induces G1 arrest and can inhibit the activity of the proliferating cell nuclear antigen (PCNA). We analyzed p21 expression during colorectal tumorigenesis, its association with its transcriptional regulator p53, and its relationship to rates of cell proliferation and apoptosis. p21 and p53 protein expression were examined in sporadic tumors and hereditary nonpolyposis colorectal cancers (HNPCCs) by immunohistochemistry (IHC) and immunoblotting. Apoptosis was examined using a DNA nick end-labeling assay, and cell proliferation was examined by PCNA staining. In normal colorectal epithelia, nuclear p21 staining was uniformly detected in crypt cells of the superficial compartment (upper one-third) that stained negatively for PCNA. p21 and PCNA expression were, therefore, mutually exclusive. In sporadic cases, a decrease in the frequency of p21 expression accompanied adenoma development and progression to carcinoma. Specifically, p21 was detected in 12 of 16 (75%) adenomas and 10 of 32 (31%) carcinomas. In contrast to sporadic cases, HNPCCs with known mutations in DNA mismatch repair genes expressed p21 in 12 of 15 (80%) carcinomas. An inverse relationship between p21 and p53 was observed wherein mutant p53 proteins were detected in 4 of 15 (27%) HNPCCs versus 22 of 32 (69%) sporadic carcinomas. Although p21+ carcinoma cells were generally negative for p53, IHC revealed that some carcinoma cells expressed both p21 and p53 proteins. Furthermore, p53-mutated SW480 colon carcinoma cells were found to coexpress p21 and p53, suggesting that p21 can also be activated by a p53-independent mechanism. No association was found between p21 or PCNA and apoptotic labeling indices in adenomas or carcinomas. In conclusion, a decrease in p21 expression accompanies neoplastic progression in sporadic cases but not in HNPCCs. This finding appears related to p53 status in that the frequency of p53 expression was significantly reduced in HNPCCs compared to sporadic cases, suggesting a difference in their molecular pathways of tumorigenesis.

Preoperative serum vascular endothelial growth factor can predict stage in colorectal cancer.

Year 1998
Kumar H. Heer K. Lee PW. Duthie GS. MacDonald AW. Greenman J. Kerin MJ. Monson JR.
University of Hull Academic Surgical Unit, Castle Hill Hospital, Cottingham, United Kingdom.
Neovascularization has been shown to be essential for the growth of solid tumors. Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. This study was conducted to determine the significance of this cytokine as a tumor marker for staging colorectal cancer. Preoperative serum VEGF was measured in 108 colorectal cancer patients and in 136 normal healthy controls. The results of this study showed a significant difference between the four T classes, Union International Contre Cancer (UICC) stages, and Dukes' stages. In comparison to serum levels in controls (median, 173.8 pg/ml), VEGF levels were significantly elevated in T2 (P = 0.003), T3, and T4 (P < 0.0005); UICC I (P = 0.001), UICC II, UICC III, and UICC IV (P < 0.0005); and Dukes' A (P = 0.001), Dukes' B, and Dukes' C (P < 0.0005). Serum VEGF showed a significant elevation over control in node-negative (P < 0.0005) and in node-positive colorectal cancer (P < 0.0005) patients. Node-positive cancer had a significant elevation of serum VEGF compared to node-negative cancer (P = 0.008). This study reveals that preoperative serum VEGF can detect all but very early colorectal cancer i.e., T1 (P = 0.06).

Schedule-dependent synergism between raltitrexed and irinotecan in human colon cancer cells in vitro.

Year 1998
Aschele C. Baldo C. Sobrero AF. Debernardis D. Bornmann WG. Bertino JR.
Department of Medical Oncology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
The quinazoline folate analogue raltitrexed (ZD1694; Tomudex) and the camptothecin derivative irinotecan are two new agents showing clinical activity against colorectal cancer. To identify the optimal conditions to achieve synergistic cytotoxicity before the clinical development of their combination, we explored the interactions between ZD1694 and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), in vitro. Cytotoxicity was evaluated with a clonogenic assay using the human colon cancer cell line HCT-8. Different schedules of administration and different dose ratios of the two agents were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27-55, 1984). Sequential short-term (1 and 4-h) exposures to SN-38 followed by ZD1694 resulted in synergistic cytotoxicity at broad dose-effect ranges. At a high level of cell kill, the synergism was greater when either equiactive doses of the two agents or higher relative doses of ZD1694 were used. A 24-h interval between exposure to SN-38 and ZD1694 significantly enhanced the magnitude of the synergy (P = 0.001). The opposite sequence or simultaneous exposures produced significantly less potentiation or nearly additive interactions (P = 0.0006 and P < 0.0001). The synergism was completely lost under conditions of more prolonged drug exposure (24-h continuous exposure). In conclusion, in this in vitro model of human colon cancer, ZD1694 and SN-38 produced synergistic cytotoxicity. Our findings support the clinical use of this combination and provide a rationale for a clinical trial using sequential short-term exposures to equiactive doses of SN-38 and ZD1694 administered sequentially with a 24-h interval.

Overexpression of stromelysin-3, BM-40/SPARC, and MET genes in human esophageal carcinoma: implications for prognosis.

Year 1998
Porte H. Triboulet JP. Kotelevets L. Carrat F. Prevot S. Nordlinger B. DiGioia Y. Wurtz A. Comoglio P. Gespach C. Chastre E.
Unite Institut National de la Sante et de la Recherche Medicale (INSERM) 482, Hopital Saint-Antoine, Paris, France.
Molecular markers can improve staging and predict aggressive clinical behavior in esophageal cancer, thus helping to define appropriate therapeutic protocols and to identify patients who will benefit from surgery. We therefore characterized, by Northern blot and/or immunohistochemistry, the relative expression of three effectors involved in the invasion, angiogenesis, and dissemination of tumor cells in esophageal cancer versus nontumoral mucosae: (a) stromelysin-3 (ST3), a member of the metalloproteinase family; (b) basement membrane 40/secreted protein acidic and rich in cysteine (BM-40/SPARC), an extracellular matrix-associated protein involved in angiogenesis; and (c) the hepatocyte growth factor receptor MET, which triggers the scattering of epithelial cells. Results were analyzed in relation to clinicopathological parameters (cpTNE) including tumor size (T), lymph node status (N), periesophageal tissue invasion (E), disease recurrence, and overall survival. The ST3, BM-40/SPARC, and MET genes were found to be overexpressed in tumor samples compared to control mucosa. BM-40/SPARC and MET mRNA levels were not linked to any one of the cpTNE, indicating that this overexpression occurs at an early stage of neoplastic progression. In contrast, ST3 expression, identified by immunohistochemistry in fibroblastic cells surrounding neoplastic islets, correlated with tumor size and periesophageal tissue invasion. Of the 36 patients studied, those with high ST3 levels had shorter disease-free survival than those with low levels, but there was no relationship between the cpTNE and disease recurrence or survival. Our study demonstrates that ST3, BM-40/SPARC, and MET are involved in different steps of esophageal carcinogenesis and that ST3 overexpression is a marker of aggressive clinical behavior. We conclude that in esophageal cancer, ST3 might help to assess survival and the risk of recurrence after surgical resection.

Biological markers as a predictor for response and prognosis of unresectable gastric cancer patients treated with 5-fluorouracil and cis-platinum.

Year 1998
Boku N. Chin K. Hosokawa K. Ohtsu A. Tajiri H. Yoshida S. Yamao T. Kondo H. Shirao K. Shimada Y. Saito D. Hasebe T. Mukai K. Seki S. Saito H. Johnston PG.
Department of Gastrointestinal Oncology, National Cancer Center Hospital East, Chiba, Japan.
We investigated the utility of examining biological markers to predict chemoresponse and survival. The subjects consisted of 39 unresectable gastric cancer patients treated with a combination of 5-fluorouracil and cis-platinum. The expression of p53, bcl-2, thymidylate synthase (TS), glutathione S-transferase pi (GST-pi), and vascular endothelial growth factor (VEGF) in the formalin-fixed biopsy samples of primary tumors before chemotherapy was examined immunohistochemically. The positive rate for VEGF, bcl-2, TS, p53, and GST-pi was 51, 10, 46, 38, and 69%, respectively. VEGF-positive cases showed a higher response rate than did negative cases (11 of 20 versus 2 of 19 cases; P = 0.0057). The cases that were negative for p53, TS, bcl-2, and GST-pi were more likely to respond to chemotherapy than the cases that were positive for these markers. The 10 cases having 4 or 5 favorable phenotypes (VEGF positive, p53 negative, bcl-2 negative, TS negative, and GST-pi negative) survived longer than the remaining 29 cases (P = 0.0069). Multivariate analysis revealed that the number of favorable phenotypes (> or = 4 versus < or = 3) had a greater impact on survival than performance status (0 versus 1 or 2), age (> 60 years versus < or = 60 years), macroscopic type (scirrhous versus nonscirrhous), histological type (intestinal versus diffuse), or tumor extent (locally advanced versus metastatic). Immunohistochemical examination of biological markers in biopsy samples may be useful in predicting the clinical outcome of unresectable gastric cancer patients treated with 5-fluorouracil and cis-platinum.

Assessment of the biological malignancy of hepatocellular carcinoma: relationship to clinicopathological factors and prognosis.

Year 1998
Mise K. Tashiro S. Yogita S. Wada D. Harada M. Fukuda Y. Miyake H. Isikawa M. Izumi K. Sano N.
First Department of Surgery, University of Tokushima School of Medicine, Japan.
It is difficult to determine the prognosis of patients with hepatocellular carcinoma (HCC). Assessment of the clinicopathological and biological malignancy of HCC may help in determining treatment strategies and predicting outcome. The tumor DNA content, p53 protein expression, proliferating cell nuclear antigen labeling index, and argyrophilic proteins of nuclear organizer regions were used as markers of biological malignancy. A correlation between these biological parameters and clinicopathological factors was sought. DNA aneuploidy was observed in 31 of 80 tumors (38.8%). Aneuploidy increased as differentiation decreased. The overall survival rate of patients with aneuploid tumors was significantly poorer than that of patients with diploid tumors. p53 overexpression was observed in 18 of 80 tumors (22.5%). The incidence of p53 positivity increased significantly with increasing tumor size and poorer differentiation. The overall survival rate of p53-positive patients was significantly worse than that of p53-negative patients. The proliferating cell nuclear antigen labeling index and the mean number of argyrophilic proteins of nuclear organizer regions were higher in more poorly differentiated lesions. We conclude that DNA ploidy and p53 expression are useful prognostic indicators in HCC. Cell proliferation increases as HCC progresses. With progression, tumors tend to become more poorly differentiated.

Motility related protein 1 (MRP1/CD9) expression in colon cancer.

Year 1998
Mori M. Mimori K. Shiraishi T. Haraguchi M. Ueo H. Barnard GF. Akiyoshi T.
Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan.
It is important to detect genes that may be good prognostic markers for colon cancer patients. With this in mind, we identified the motility related protein-1 (MRP1/CD9) gene in human colon tissues. The aim of this study was to clarify the significance of MRP1/CD9 gene expression in human colon cancers. We performed the differential mRNA display technique between tumor/normal paired samples of the colon and identified MRP1/CD9. Eighty-two surgical specimens of primary colorectal cancer were analyzed by means of reverse transcription-PCR for the MRP1/CD9 gene. Its expression status and clinicopathological variables were analyzed univariately and multivariately. The MRP1/ CD9 mRNA expression was positive in 56 cases and negative in 26 cases. The MRP1/CD9 negative cases showed a significantly higher frequency of venous-vessel invasion and liver metastasis, or a worse prognosis than the MRP1/CD9 positive cases (P < 0.05). Multivariate analysis with the Cox regression model disclosed that MRP1/CD9 expression was an independent prognostic factor distinct from the lymph node status. The findings imply that the study of MRP1/CD9 expression may be useful for predicting prognosis of patients with colorectal cancer.

Prognostic values of cathepsin B and carcinoembryonic antigen in sera of patients with colorectal cancer.

Year 1998
Kos J. Nielsen HJ. Krasovec M. Christensen IJ. Cimerman N. Stephens RW. Brunner N.
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
The level of cathepsin B (Cat B) was determined in sera obtained preoperatively from 325 patients with colorectal cancer using an ELISA. Control sera from 90 healthy blood donors were analyzed. The levels of Cat B detected included all forms that were present in the sera, i.e., mature enzyme, precursor molecule, and enzyme-inhibitor complexes. The level of Cat B was significantly increased in sera of patients with colorectal cancer. The median level was 10.7 ng/ml versus 2.1 ng/ml in controls (P < 0.0001). A correlation between Cat B serum level and advanced Dukes' stage (P < 0.003) was found, whereas no associations have been found with age, sex, or level of carcinoembryonic antigen (CEA). In survival analysis, the patients with high serum Cat B experienced significantly lower survival probability. At the optimal cutoff value of 9.4 ng/ml, the relative hazard ratio was 1.8 (95% confidence interval, 1.1-2.8; P = 0.016) in the univariate Cox proportional hazards model. The median observation time was 4.4 years (range, 3.2-5.5 years). In multivariate analysis, Dukes' stage was the strongest prognostic variable, followed by age, whereas serum Cat B and CEA were not significant prognostic factors in this model, in accordance with their association with Dukes' stage. When the data for Cat B and CEA were combined, CEA-positive patients were further separated by Cat B into high- and low-risk groups. Patients with high serum levels of both molecules had significantly shorter survival (relative hazard ratio of 2.2; 95% confidence interval, 1.5-3.2; P < 0.0001), as compared with patients with low levels of both molecules.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features.

Year 1998
Yamada T. Nakamori S. Ohzato H. Oshima S. Aoki T. Higaki N. Sugimoto K. Akagi K. Fujiwara Y. Nishisho I. Sakon M. Gotoh M. Monden M.
Department of Surgery II, Osaka University Medical School, Suita City, Japan. tyamada@surg2.med.osaka-u.ac.jp
We investigated the presence of K-ras gene mutation in plasma DNA and assessed its clinical value in patients with pancreatic adenocarcinoma. Mutations in codon 12 of the K-ras gene were examined by mutant allele-specific amplification method using DNA extracted from surgical specimens and plasma samples of 21 patients with pancreatic adenocarcinoma. K-ras gene mutation was detected in 15 of 21 (71%) primary tumors. In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. None of four patients with chronic pancreatitis or five healthy subjects had such mutations in plasma DNA. Tumors positive for K-ras gene mutation in plasma DNA were significantly larger (P = 0.04) and less likely to result in a curative cure after surgical resection (P = 0.09) than those negative for the mutation. Other clinicopathological features, including age, sex, histological type, mode of invasion, and metastasis, did not correlate with K-ras gene mutations in plasma DNA. Treatment resulted in disappearance of K-ras gene mutations in plasma DNA in six of nine (67%) patients. Three patients with a persistently positive K-ras gene mutation in pre- and post-treatment plasma samples were likely to show early recurrence or have a progressive disease. Our findings suggest that K-ras gene mutation can be detected in plasma DNA of patients with pancreatic adenocarcinoma. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment.