Characterization of monoclonal antibodies directed against squamous cell carcinoma antigens: report of the TD-10 Workshop.

Thirteen monoclonal antibodies directed against squamous cell carcinoma antigens (SCCA1 and SCCA2) were obtained from five international collaborating laboratories participating in the ISOBM TD-10 Workshop. Native and recombinant forms of SCCA were used in a wide variety of approaches to determine the reactivity and specificity of these antibodies. Based on reactivity, the antibodies could be divided into three groups: the SCCA1-reactive group containing those that reacted only with recombinant SCCA1 (rSCCA1) and native SCCA1 (nSCCA1) antigens, the SCCA2-reactive group containing those that reacted only with recombinant SCCA2 (rSCCA2), and the pan-reactive group containing those antibodies that reacted with rSCCA1, nSCCA1, and rSCCA2. Binding to radioiodinated rSCCA1 showed that all reactive antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Binding to labelled rSCCA2 demonstrated that five antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Antibody reactivity on Western blots was tested with nonreduced and reduced native and recombinant SCCA1 and SCCA2. In general, these findings showed that reduction had little effect on binding to SCCA1, but often a strong effect on the binding to SCCA2. Binding of antibodies to rSCCA1 and rSCCA2 in complexes with cathepsin L and G, respectively, was used to assist in the localization of epitope regions in enzyme-complexed SCCA. Cross-inhibition experiments showed that SCCA1-reactive antibodies represent two different epitope groups, and this is supported by their ability to make SCCA1-specific assays by combining antibodies from the two epitope groups. The SCCA2-reactive group represents two related antibodies and one unique as seen in cross-inhibition, but they do not form successful assay combinations. Classification of the pan-reactive antibodies is more difficult, as some epitope groups differ when results from rSCCA1 are compared with rSCCA2 as the target. However, two antibodies are outstanding, SCC107 and SCC113, as they are high-affinity antibodies which react equally well with free and protease complexes of SCCA1 and SCCA2. The precise location of epitopes was further studied using sequential overlapping peptides and homology modelling. The findings from this workshop strongly indicate that the recombinant antigens (rSCCA1 and rSCCA2) are very similar in epitope structure to the native counterparts in saliva, and squamous epithelium from normal and cancer tissues. Therefore, it is reasonable to conclude that the specificities found are reliable and have application for antibody measurement of all forms of squamous cell carcinoma in serum except SCCA2 in complex with its protease.

[Subjective criteria of chest CT findings for predicting pathological features and postoperative outcomes of small peripheral lung cancer (< or = 2 cm)].

One hundred fifty five patients with completely resected peripheral non-small cell lung cancer, clinically diagnosed 2 cm or less in diameter, are retrospectively reviewed on their preoperative chest CT films, clinico-pathological features, and postoperative outcomes. Pathologic type was classified according to Noguchi's classification. 7% and 8% of all the patients had pathologic N 1 and N 2 diseases, respectively. 19% of all the patients undergone limited resection (segmentectomy or partial resection). Maximum area of the tumor/soft tissue density area of the tumor (M/S ratio) was manually measured by chest CT film. According to the logistic regression analysis, M/S ratio was the only predicting factor of regional lymph node metastasis among factors including pleural indentation, spiculation, and maximum area of the tumor. Univariate analysis showed that maximum area of the tumor, pleural indentation, and M/S ratio were the significant factor for postoperative disease free survival. According to multivariate analysis of postoperative disease free survival with adjustment for operative modality, the result was same as that of univariate analysis. In conclusion, our determined criteria of the chest CT accurately predicted pathological status and postoperative outcome of patients with small peripheral lung cancer. These factors would be useful for stratification factor of prospective clinical study.

De novo uterine sarcoma with good response to neo-adjuvant chemotherapy.

We report here the extremely rare case of a 28-year-old woman with advanced stage uterine sarcoma arising soon after a cesarean section. She underwent an abdominal cesarean section because of a breech presentation. At the time of the procedure, there were no abnormal findings such as leiomyoma of the uterus in the abdominal cavity. One year later, she was referred to our hospital because of a large abdominal tumor. Transabdominal power Doppler ultrasonography and magnetic resonance imaging (MRI) showed a large hypervascular tumor in the abdominal cavity. Her serum levels, for the two tumor markers carbohydrate antigen CA125 and LDH, were elevated, at 219 U/ml (< 35 U/ml) and 862 IU/l (115 U/ml-217 U/ml), respectively. On the basis of a diagnosis of malignant tumor of gynecological origin, exploratory laparotomy was performed, and through biopsy, the tumor was found to be advanced undifferentiated uterine sarcoma. She exhibited a good response to neoadjuvant chemotherapy consisting of cisplatin, epirubicin, and dimethyltriazenoimidazole carboxamide (DTIC) every 28 days, which was successfully followed by a hysterectomy.

Immature teratoma with gliomatosis peritonei associated with pregnancy.

Immature teratoma, which contains variable quantities of immature tissues that resemble those of the embryo, is one of the primitive germ cell tumors. It occurs most frequently in young women but it is rarely reported in association with pregnancy. We report a case of immature teratoma associated with pregnancy exhibiting unique MR findings with pathologic correlation.

Electrophoretic characterization of heat-stable squamous cell carcinoma antigen.

The aim of this study was to investigate the heat stability of squamous cell carcinoma (SCC) antigen, a tumor-associated serine proteinase inhibitor (serpin), in tumor tissue extract by electrophoretic methods. After heat treatment at 70 degrees C for 2 h, the tumor tissue extract showed a single main protein band of 45 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which reacted with a monoclonal antibody specific for SCC antigen. The heat-stable SCC antigen was separated by two-dimensional electrophoresis (2-DE) into four spots with pI 6.4-5.9 and Mr 44500-45 000 of SCC antigen-1. Furthermore, the SCC antigen-1 still showed its inhibitory activity against a cysteine proteinase, papain, by gelatin zymography. These results suggest that heat treatment of protein sample at 70 degrees C for 2 h may be a useful method for a partial purification of SCC antigen-1 which can inhibit lysosomal cysteine proteinases such as cathepsin L, S, and K.

Novel forms of squamous cell carcinoma antigen transcripts produced by alternative splicing.

Squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin serine protease inhibitor family, and the serum level of SCCA is a tumor marker of squamous cell carcinoma. Reverse transcription (RT)-PCR of the squamous cell carcinoma cell line showed the existence of a 156 base shorter transcript compared with that of SCCA1 cDNA. By inverse PCR, we cloned the full length cDNA of this SCCA (SCCA1b). Sequence analysis of the complete 1541 bp SCCA1b cDNA showed that it coded for 338 amino acids and had no typical signal sequence in the NH(2) terminus. The cDNA was expressed in Escherichia coli and the product was detected using Western blotting with antibodies against SCCA. Furthermore, RT-PCR of the full coding region of SCCA2 cDNA from cancer tissue showed the existence of a 63 base short transcript (SCCA2b). A comparison of SCCA1b and SCCA2b cDNA with the SCCA1 and SCCA2 genes showed that these messages were derived from each gene by an alternative splicing mechanism.

Suppression of a squamous cell carcinoma (SCC)-related serpin, SCC antigen, inhibits tumor growth with increased intratumor infiltration of natural killer cells.

Squamous cell carcinoma (SCC) antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, serves as a circulating marker of squamous cell carcinoma (SC). One of the SCCAs, SCCA1, has been suggested to play a role in the attenuation of apoptosis in vitro and in the augmentation of tumor growth in vivo. In the present study, the infection of a SCC cell line (SKG IIIa) with recombinant retrovirus that expressed the antisense SCCA mRNA suppressed expression of SCCA in vitro. Local administration of this retrovirus into tumors by inoculation in nude mice suppressed tumor growth. Treatment of tumor tissue in vivo is also associated with increased numbers of apoptotic tumor cells and large mononuclear cells in the tumor. To test the possible role of SCCA in the infiltration of large mononuclear cells, we analyzed the effect of SCCA1 on migration of natural killer (NK) cells induced by monocyte-chemoattractant protein-1 in vitro. SCCA1 suppressed migration of NK cells completely, and this inhibitory effect was lost by mutation of the reactive site loop of SCCA1. These results suggest that antisense SCCA may suppress the growth of SCC in vivo not only by the augmentation of intracellular apoptosis but also by the increased infiltration of NK cells into the tumor.

Serum anti-p53 antibodies in uterine and ovarian cancer: association with dna sequence copy number abnormalities.

The aim of the present study was to evaluate the clinical significance of the serum anti-p53 antibody in patients with uterine and ovarian cancer. Some of the ovarian patients were also evaluated for overexpression of p53 by immunohistochemistry and for cytogenetic alterations by comparative genomic hybridization (CGH). Serum anti-p53 antibodies were determined by an enzyme immunoassay kit. The antibody was detected in 8/30 (27%) of ovarian cancers, in 12/86 (14%) cancers of the uterine cervix, in 5/41 (12%) cancers of the uterine body, and 0/9 (0%) healthy women. The overall survival rate in patients with ovarian cancer was significantly worse in patients with anti-p53 antibody positivity than that in patients with anti-p53-antibody-negative cancers using the log rank test (p = 0.017). There was a significant correlation between the presence of anti-p53 antibody and tissue overexpression of p53 in ovarian cancers. CGH analysis showed that the aberrations in DNA sequence copy number in ovarian cancers were significantly increased in anti-p53-antibody-positive cases compared to antip53-antibody-negative cases including increased copy number on 20q and reduced copy number on 5q and 13q. Although the exact relationship between the presence of serum anti-p53 antibody (specific humoral response) and cytogenetic alterations is still unknown, these findings suggest that the measurement of serum anti-p53 antibody may be useful for the assessment of genetic instability and tumor biological aggressiveness.

Squamous cell carcinoma antigen suppresses radiation-induced cell death.

Previous study has demonstrated that squamous cell carcinoma antigen (SCCA) 1 attenuates apoptosis induced by TNF alpha, NK cell or anticancer drug. In this study, we have examined the effect of SCCA2, which is highly homologous to SCCA1, but has different target specificity, against radiation-induced apoptosis, together with that of SCCA1. We demonstrated that cell death induced by radiation treatment was remarkably suppressed not only in SCCA1 cDNA-transfected cells, but also in SCCA2 cDNA-transfected cells. In these transfectants, caspase 3 activity and the expression of activated caspase 9 after radiation treatment were suppressed. Furthermore, the expression level of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was suppressed compared to that of the control cells. The expression level of upstream stimulator of p38 MAPK, phosphorylated MKK3/MKK6, was also suppressed in the radiation-treated cells. Thus, both SCCA1 and SCCA2 may contribute to survival of the squamous cells from radiation-induced apoptosis by regulating p38 MAPK pathway.

Nondenaturing two-dimensional electrophoretic analysis of loop-sheet polymerization of serpin, squamous cell carcinoma antigen-2.

Two homologous serine proteinase inhibitors (serpins), squamous cell carcinoma (SCC) antigen-1 and -2 were separated by nondenaturing two-dimensional electrophoresis combined with immunostaining to acquire further information on these proteins under physiological conditions. Polymers of SCC antigen-2 were detected in cytosolic extracts prepared from tumor tissues. The polymer formation of SCC antigen-2 was apparently decreased and the SCC antigen-2-synthetic peptide binary complexes were newly formed by the addition of synthetic peptide with sequences corresponding to residues from P14 to P2 in the reactive center loop of SCC antigen-2. On the other hand, the incubation with synthetic peptides having the sequence of the reactive center loop of SCC antigen-1 or antithrombin had no effect on polymerization of SCC antigen-2. These data suggest that the polymerization of SCC antigen-2 may occur spontaneously in vivo by the loop-sheet mechanism of serpin.

Specific detection and quantitation of SCC antigen 1 and SCC antigen 2 mRNAs by fluorescence-based asymmetric semi-nested reverse transcription PCR.

Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous epithelia and malignant squamous cell tissues. The serum level of SCCA has been used to evaluate treatment efficacy, clinical course of disease, and recurrence. SCCA is produced by at least two genes (SCCA1 and SCCA2); both of them have been located on chromosome 18q21.3. It has been difficult to examine the expression levels of SCCA1 and SCCA2 mRNAs separately because of their high homology at nucleotide level. In the present study, asymmetric semi-nested reverse transcription PCR, based on the principle of fluorescence energy transfer, enabled to quantitate the copy numbers of both SCCA1 and SCCA2 mRNAs. Using this method, the expression levels of these mRNAs were evaluated in normal and malignant squamous tissues. The copy number of SCCA2 mRNA was higher in malignant tissues than in normal tissues, while those of SCCA1 mRNA did not significantly differ between normal and malignant tissues. These data indicate that specific quantitation of the expression level of SCCA2 mRNA may be useful for the diagnosis and management of patients with squamous cell carcinoma.

Involvement of reactive oxygen species and nitric oxide in gastric ischemia-reperfusion injury in rats: protective effect of tetrahydrobiopterin.

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), a cofactor of nitric oxide (NO) synthase, attenuates gastric ischemia-reperfusion injury induced by clamping of the celiac artery. Gastric injury was assessed by a formation of gastric mucosal erosions. The gastric injury was observed at 30 and 60 min after reperfusion following 30-min ischemia and was reduced by superoxide dismutase (SOD), catalase, or NO synthase inhibitors. Therefore, reactive oxygen species (ROS) and NO seem to be implicated in the ischemia-reperfusion injury. Treatment with BH4 reduced the ischemia-reperfusion injury. Pretreatment with sepiapterin, a precursor of BH4, also reduced the ischemia-reperfusion injury with an increase in BH4 content in serum and stomach. Both the increase in BH4 content and the protective effect of sepiapterin were prevented of pretreatment with N-acetylserotonin, an inhibitor of BH4 synthesis. These results suggest that the increase in BH4 content may protect against gastric ischemia-reperfusion injury via reduction of ROS and/or NO toxicity. BH4 might be useful as a therapeutic agent for gastric ischemia-reperfusion injury.

The long-term evaluation of pulmonary toxicity following isolated lung perfusion with melphalan in the rat.

The present study was conducted to evaluate the long-term pulmonary toxicity of isolated lung perfusion (ILP) with melphalan in the rat model. F344 rats were treated by ILP with 1 mg of melphalan or buffered hespan (BHE). The rats in the melphalan group were sacrificed randomly 30, 60, 90, 120, 150, and 180 days after the perfusion. Pulmonary toxicity was evaluated by pathological analysis. In the melphalan group, light and electron microscopic findings revealed perivascular and peribronchial edema, and septal thickening with cellular infiltration of the interstitial space 30 days after the perfusion, but all of these changes had disappeared by 60 days. Azan stain showed a slight increase of the connective tissue at the alveolar wall in the melphalan group, but no progressive pulmonary interstitial fibrosis was observed after 180 days. Transmission electron microscopy showed minimal proliferation of the type II pneumocytes of normal appearance in the melphalan group. In conclusion, the long-term pulmonary toxicity of ILP with melphalan is acceptable; however clinical trials of this therapy need to be conducted.

Identification of squamous cell carcinoma antigen-2 in tumor tissue by two-dimensional electrophoresis.

The aim of this study was to identify two homologous serine proteinase inhibitor (serpin) molecules, squamous cell carcinoma (SCC) antigen-1 and -2, by two-dimensional electrophoresis (2-DE), combined with immunoblotting, and examine their expression in tumor tissue. The recombinant SCC (rSCC) antigen-1 showed four spots with p/ 6.5, 6.4, 6.3 and 6.0, whereas rSCC antigen-2 showed a more acidic spot with p/5.95. SCC antigen in tumor tissue appeared in three new acidic spots (p/5.7-5.5, M(r) 44 500), numbered 5, 6 and 7, besides the previously reported four spots numbered 1 to 4. These new acidic spots of SCC antigen apparently increased in SCC tissue. Treatment of tissue extract by carboxymethyl (CM)-papain agarose matrix extinguished spots 1 to 4 encoded on the SCCA1 gene, but not 5 to 7 on the SCCA2 gene. Overexpression of the SCCA2 gene may play an important role in the malignant behavior of tumor cells.

Biological role of SCC antigen.

SCC antigen is a tumor-associated protein of squamous cell carcinoma of various organs. So far, two genes (SCC Ag-1 and SCC Ag-2) have been identified, and their products are highly homologous and classified as serine protease inhibitors (serpin). Recombinant SCC antigen-1 inhibits chymotrypsin and cathepsin L in vitro, indicating that it is inhibitory type serpin. Transduction of tumor cells with SCC antigen-1 reveals that SCC antigen-1 inhibits apoptosis of tumor cells induced by anticancer drug, TNFalpha or NK cells. Therefore SCC antigen-1 may work in cancer cells for tumor growth, and in normal squamous epithelium for differentiation by means of the inhibition of apoptosis. Recombinant SCC antigen-2 inhibits cathepsin G and mast cell chymase, suggesting that it protects epithelial cells from the inflammation induced by these proteases.

Surgery for abdominal aortic aneurysms associated with malignancy.

Of 148 patients treated for abdominal aortic aneurysms (AAA), 33 (22%) also had cancer. According to the classification of Szilagyi, there were 13 patients in group I, 19 in group II, and 1 in group IV. In group I, the mean interval between the cancer and AAA operations was 7 years (range 1-14 years). Aneurysmectomy was performed in 9 patients, wrapping in 2, and no operation in 2. In group II, a two-stage operation was performed in 8 patients, a single-stage operation in 4, only surgery for cancer in 4, and no operation in 3. Of 4 patients undergoing single-stage operations, 3 had colorectal cancer, and there were no postoperative complications such as graft infection or anastomotic breakdown. In group I, 6 of 13 patients died, but there were no cancer deaths. In group II, 9 of 19 patients died, 6 from progressive cancer. The group IV patient also died of cancer. These results suggest that if a patient can tolerate surgery for both diseases, a single-stage operation is preferable.

Cost analysis for thoracoscopy: thoracoscopic wedge resection and lobectomy.

We reviewed our experience with video-assisted thoracic surgery (VATS) in our most recent 80 patients for the purpose of cost analysis. The costs incurred in the patients undergoing a VATS wedge resection for nodules (n = 30) and a VATS lobectomy for lung cancer (n = 10) were compared with the costs in similar patients undergoing a wedge resection (n = 20) and lobectomy (n = 20) using open techniques. The disposable instrument costs were US $1071 higher for a VATS wedge resection; however, the operative time was shorter (0.99 h for VATS versus 1.75 h for the open procedure). The length of hospital stay was also shorter after a VATS wedge resection (10.4 days for VATS versus 16.8 days for the open procedure), thus resulting in lower total hospital charge in the VATS group. The disposable instrument costs were $3190 higher for a VATS lobectomy, and the operative time was longer (5.56 h for VATS versus 4.25 h for the open procedure). The length of hospital stay was similar in both groups (25.2 days for VATS versus 27.7 days for the open procedure), thus resulting in a higher total hospital charge in the VATS lobectomy group. The cost of a VATS wedge resection for removing peripheral nodules is competitive with that of open techniques, but the cost of a VATS lobectomy is higher than that for an open lobectomy.

Dissecting aortic aneurysm involving an anomalous right subclavian artery and isolated left vertebral artery: case report and review of the literature.

A 54-year-old hypertensive woman was admitted with severe interscapular back pain. A chest radiograph showed marked widening of the mediastinum. Aortography demonstrated a DeBakey type III, a thoracic aortic dissection and an anomalous right subclavian artery which was associated with an isolated left vertebral artery. The patient underwent aortic arch replacement with 5 branches and made an uneventful recovery. As far as we can determine, this is the first reported occurrence of these anomalies together with acquired disease of the aorta.

Isolated lung perfusion with cisplatin in a rat lung solitary tumor nodule model.

BACKGROUND: The present study was conducted to evaluate the toxicity, pharmacokinetics and anti-tumor potency of isolated lung perfusion (ILP) with cisplatin in a visible lung tumor nodule model in rats. MATERIALS AND METHODS: A solitary tumor nodule was established by the injection of Methylcholanthrene-induced sarcoma cells into the left lung. Thirty rats were randomized to undergo ILP with either 0.1, 0.25, or 0.5 mg/mL cisplatin and buffered hespan (BHE), or with an intravenous injection of 1.0 or 2.5 mg cisplatin. RESULTS: The highest dose of cisplatin tolerated by the rats was 0.1 mg/mL for perfusion. A much higher platinum concentration in the tumor, of 6.67 +/- 1.64 vs. 2.51 +/- 0.60 micrograms/g tissue, but a significantly lower concentration in the serum and kidneys, was achieved by perfusion compared to that achieved by intravenous injection. A significantly lower tumor weight and 20% complete treatment response was achieved in rats given cisplatin than in those given BHE perfusion at 43.9 +/- 11.6 vs. 226.3 +/- 44.6 mg. CONCLUSION: ILP with cisplatin achieved superior results to intravenous injection according to the levels of toxicity and pharmacokinetic analysis, and it was effective against a visible tumor nodule model in rats.

Isolated lung perfusion with doxorubicin prolongs survival in a rodent model of pulmonary metastases.

BACKGROUND: We developed a rodent model of unilateral pulmonary metastases to evaluate long-term survival after isolated lung perfusion with doxorubicin. METHODS: In the model development study, on day 0, two groups of F344 rats (n = 15) underwent transient right pulmonary artery occlusion for either 5 or 10 minutes at the time of intravenous injection of methylcholantrene-induced sarcoma cells. On day 14, all animals were sacrificed and lung nodules counted. In the survival study, on day 0, 21 rats received intravenous injection of sarcoma cells with concomitant 10-minute right pulmonary artery occlusion. On day 7, eight rats underwent left isolated lung perfusion with doxorubicin (6.4 mg/kg); five rats underwent perfusion with buffered Hespan; six untreated rats were studied as controls. RESULTS: Ten of fifteen animals (67%) in the model study with 5-minute pulmonary artery occlusion had right-sided tumor nodules. Ten-minute occlusion resulted in a tumor-free right lung in all animals. In the survival study, all animals in the Hespan and control groups died of massive tumor replacement of the left lung, with median survival times of 20 and 18 days, respectively. The median survival time of 36 days for the animals undergoing isolated lung perfusion with doxorubicin was significantly longer (p < 0.00001). The left lung of two of the doxorubicin perfused rats was tumor-free at 6 weeks. CONCLUSIONS: Isolated lung perfusion with doxorubicin results in a durable response and prolongs survival in the treatment of experimental sarcoma pulmonary metastases.