Immunocytochemical detection of disseminated tumor cells in the bone marrow of patients with esophageal carcinoma.

BACKGROUND: Approximately half of the patients diagnosed with localized esophageal cancer die of metastatic disease within the first 2 years following tumor resection. The development of monoclonal antibodies (MAbs) directed against epithelial cell-associated and tumor antigens has enabled the detection of single disseminated tumor cells in secondary organs. PURPOSE: We used MAbs directed against epithelial cell antigens (i.e., cytokeratins) to determine the proportion of patients with esophageal cancer who display isolated tumor cells in their bone marrow. In addition, we evaluated the prognostic significance of a finding of bone marrow tumor cells in patients with esophageal cancer whose tumors were completely resected. METHODS: Prior to the initiation of treatment, bone marrow was aspirated from both sides of the upper iliac crests of 90 patients with squamous cell carcinoma of the esophagus. Bone marrow was also obtained from a population of 30 individuals who had not been diagnosed with cancer. Tumor cells in cytologic bone marrow preparations were detected by use of an assay that employed the MAbs CK2 (directed against cytokeratin 18), KL1 (directed against a 56,000-kd pan-cytokeratin component), and A45-B/B3 (directed against an epitope common to cytokeratins 8, 18, and 19) plus the alkaline phosphatase anti-alkaline phosphatasestaining method. Bone marrow biopsies, for conventional histologic examination with Giemsa staining, were performed on 62 patients. The Kaplan-Meier method and the logrank test were used to assess disease-free and overall survival according to the presence or absence of tumor cells in the bone marrow of 42 patients with completely resected tumors. Reported P values are two-sided. RESULTS: Cytokeratin-positive tumor cells were detected in the bone marrow of 37 (41.1%) of the 90 total patients. The number of tumor cells detected per 10(5) mononuclear bone marrow cells ranged from one to 82. No significant differences in the numbers of disseminated tumor cells were noted for patients diagnosed with tumors at different stages. Only two (3.2%) of 62 bone marrow specimens examined after Giemsa staining showed morphologically identifiable tumor cells. Tumor cells were not detected in the bone marrow of patients who had not been diagnosed with cancer. After a median follow-up of 15.5 months (range, 6-33 months), 15 (79.0%) of 19 patients with completely resected tumors and tumor cells in their bone marrow had relapses compared with three (13.0%) of 23 patients with completely resected tumors and no tumor cells in their bone marrow (P = .019, logrank test). Patients with completely resected tumors and tumor cells in their bone marrow had significantly shorter overall survival than corresponding patients without tumor cells in their bone marrow (P = .036, logrank test). CONCLUSIONS AND IMPLICATIONS: Dissemination of esophageal cancer cells to the bone marrow is more frequent than expected from the rare occurrence of overt skeletal metastases. In general, the presence of tumor cells in the bone marrow may be an indicator of the disseminatory potential of individual tumors.

E-cadherin gene mutations provide clues to diffuse type gastric carcinomas.

The calcium-dependent homophilic cell adhesion molecule and candidate suppressor gene, E (epithelial)-cadherin, plays a major role in the organization and integrity of most epithelial tissues. Diffusely growing gastric carcinomas show markedly reduced homophilic cell-to-cell interactions. We speculated that mutations in the E-cadherin gene may be responsible for the scattered phenotype of this type of carcinoma. For that reason we have examined E-cadherin in 26 diffuse type, 20 intestinal type and 7 mixed gastric carcinomas (Laurén's classification) at the DNA, RNA, and protein levels. Reverse transcription polymerase chain reaction and direct sequencing of amplified E-cadherin complementary DNA fragments revealed inframe skipping of either exon 8 or exon 9 in 10 patients with diffuse tumors and an exon 9 deletion in one patient with a mixed carcinoma; both exons encode putative calcium binding domains. These alterations were not seen in nontumorous gastric tissues. Splice site mutations responsible for the exon deletions were identified in six of these patients, eliminating the possibility of alternative splicing mechanisms. Five of these splice site alterations were confirmed as somatic mutations. Non-splice site mutations were observed in three diffuse type tumors, namely a 69-base pair deletion of exon 10 and two point mutations, one of which destroys a putative calcium binding region. Immunohistochemical evaluation showed E-cadherin immunoreactivity in tumors and lymph node metastases of patients expressing abnormal mRNA. The allelic status of the E-cadherin gene was analyzed in one patient, revealing loss of heterozygosity with retention of a mutated E-cadherin allele. Overall, E-cadherin mutations were identified in 50% (13 of 26) of the diffuse type and in 14% (1 of 7) of the mixed carcinomas. In contrast, two silent E-cadherin mutations (not changing the amino acid sequence) were detected in two tumors of the intestinal type. Our study provides strong in vivo evidence that E-cadherin gene mutations may contribute to the development of diffusely growing gastric carcinomas and support a tumor/metastasis suppressor gene hypothesis.

Prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in completely resected gastric cancer.

The prognostic impact of the proteolytic factors urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) was evaluated in 76 completely resected gastric cancer patients enrolled in a prospective study. All patients underwent macroscopically and microscopically residual tumor-free resection (category R0, Union International Contre Cancer, 1987). uPA and PAI-1 levels were quantified in detergent-extracted (Triton X-100) specimens of primary gastric tumors by enzyme-linked immunosorbent assays. Median values of 1.57 ng uPA/mg protein were determined in tumor tissue extracts compared to 0.14 ng uPA/mg protein in normal mucosa. For PAI-1, 0.93 ng PAI-1/mg protein versus 0.09 ng PAI-1/mg protein was calculated. uPA levels in tumor tissue extracts were significantly correlated with vascular invasion, Laurén classification, and WHO classification, whereas PAI-1 levels showed a significant correlation with advanced lymph node involvement, depth of invasion, tumor stage, site of tumor, and the Laurén, Borrmann, and WHO classifications. Elevated uPA and PAI-1 levels were found to be associated with poor prognosis. The optimal cutoff values indicating a group of patients with shorter survival were 1.5 ng uPA/mg protein and 1.25 ng PAI-1/mg protein, respectively (Classification and Regression Tree analysis). Patients with either high uPA or PAI-1 values were significantly associated with decreased survival (median time of survival was 23 months (high) versus 44 months (low). By univariate Cox regression analysis, it was shown that TNM categories, WHO classification, size of tumor, uPA and PAI-1 levels were all significantly associated with survival. However, in multivariate Cox regression analysis of these grouped variables, nodal status, PAI-1 levels, and WHO classification were the only independent prognostic factors. The relative risks of failure were 5-, 2.9-, and 2.4-fold, respectively. We conclude that PAI-1 and uPA positivity may serve as new prognostic factors in gastric cancer, predicting shorter survival even in clinically important subgroups of patients.

Strong prognostic impact of tumor-associated urokinase-type plasminogen activator in completely resected adenocarcinoma of the esophagus.

The serine protease system has been shown to play an important role in the invasive potential of a variety of tumors. To date, however, there are little data about the expression of these proteases in esophageal carcinoma. To determine the level of expression and the significance of urokinase-type plasminogen activator (uPA) and its plasminogen activator inhibitor type 1 (PAI-1) in adenocarcinoma of the esophagus, we studied 54 tumor cases and a control group of normal gastric mucosa cases with ELISA using detergent-extracted samples. uPA and PAI-1 were significantly elevated as compared to control tissue by factors of 16 and 14, respectively. Median levels of both uPA and PAI-1 showed significant correlation with tumor pT, pN, and pM categories, whereas the presence of lymphatic invasion correlated only with the uPA content and tumor grade correlated only with PAI-1 content. Using maximally selected statistics, a cutoff value was found for uPA (2.85 ng/mg protein) but not for PAI-1, which divided the study group into significantly poorer and better survival subgroups. By univariate analysis, depth of tumor invasion (pT), lymph node involvement (pN), number of involved lymph nodes, lymph node ratio, distant nodal metastases [pM1(Iym)], lymphatic invasion, and uPA showed significant correlations with patient survival. By multivariate analysis, uPA (first rank), pN, and pM (lym) were identified as independent prognostic factors, with relative risks of 8.4, 4.1, and 4.3, respectively. In a second survival analysis method, a prognostic model was developed using classification and regression trees analysis, in which a significant difference among three patient survival groups was distinguished using the variables "number of involved lymph nodes" and "uPA content." In summary, tumor uPA content as determined by ELISA appears to be a powerful, independent prognostic factor for survival in adenocarcinoma of the esophagus.

Preclinical activity of taxotere (RP 56976, NSC 628503) against freshly explanted clonogenic human tumour cells: comparison with taxol and conventional antineoplastic agents.

Taxotere (TER) and taxol (TA) are new antitumour agents currently undergoing clinical evaluation. We studied the antineoplastic effects of these agents (final concentrations: 4.0, 0.4, 0.04 mumol/l) on the in vitro proliferation of clonogenic cells from freshly explanted human tumours using a capillary soft agar cloning system. We also compared the activity of these new compounds to conventional antineoplastic agents (bleomycin, cisplatin, dacarbazine, doxorubicin, etoposide, 5-fluorouracil, vinblastine, interferon-alpha 2). Using a 21-28-day continuous drug exposure, 54/81 specimens (67%) were evaluable for comparisons, and using a 1-h drug exposure followed by 21-28 days incubation, 50/80 specimens (63%) were similarly evaluable. With both schedules, TA and TER showed concentration-related antitumour activity. At 0.4 mumol/l, median colony survival was 0.61 x control (range 0.09-0.96) for TA and 0.51 x control (0.15-0.81) for TER in the 1-h incubation (P = 0.0002). Median colony formation was also reduced significantly more by TER as compared to TA in the long-term incubation schedule. Statistical analysis indicated that TER but not TA was significantly more active than cisplatin (P = 0.02), doxorubicin (P = 0.01), 5-fluorouracil (P = 0.01) and interferon-alpha 2 (P = 0.01). We conclude that TER and TA are more active against in vitro tumour colony formation from freshly explanted human tumours. TER appears to be slightly more active than taxol and promises to be active against tumours resistant to conventional antineoplastics.

Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol.

Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.

CD87-positive tumor cells in bone marrow aspirates identified by confocal laser scanning fluorescence microscopy.

Dissemination of single tumor cells to the bone marrow is a common event in cancer. The clinical significance of cytokeratin-positive cells detected in the bone marrow of cancer patients is still a matter of debate. In gastric cancer, overexpression of the receptor (uPAR or CD87) for the serine protease urokinase-type plasminogen activator (uPA) in disseminated cancer cells indicates shorter survival of cancer patients. A new immunofluorescence approach, applying confocal laser scanning microscopy, is introduced to locate CD87 antigen in cytokeratin-positive tumor cells and to quantify the CD87 antigen by consecutive scanning. At first, cytokeratin 8/18/19-positive carcinoma cells are identified at excitation wavelength 488 nm using monoclonal antibody A45B/B3 to the cytokeratins and goat anti-mouse IgG labeled with the fluorochrome Alexa488. Next, CD87 in tumor cells is identified by chicken antibody HU277 to the uPA-receptor and goat anti-chicken IgY labeled with fluorochrome Alexa568 (excitation wavelength 568 nm) and the fluorescence signal quantified on a single cell basis using fluorescently labeled latex beads as the fluorescence reference. From 16 patients with gastric or esophageal carcinoma, bone marrow aspirates were obtained, stained for cytokeratins and CD87 and then subjected to laser scanning fluorescence microscopy. Three of six gastric cancer patients had tumor cells present in the bone marrow of which 2 stained for CD87. Three of ten esophageal carcinoma patients had tumor cells in the bone marrow, all three samples stained for CD87. CD87-positive tumor cells were also dissected from stained bone marrow aspirates by laser microdissection microscope to allow analysis of single cells at the gene level.

Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesothelial.

A new monoclonal antibody, Ber-EP4, directed against a partially formol resistant epitope on the protein moiety of two 34 kilodalton and 39 kilodalton glycopolypeptides on human epithelial cells is described. Immunostaining of a wide range of normal and neoplastic human tissues and cell lines showed that all carcinomas and all non-neoplastic epithelial cells, except hepatocytes, parietal cells, and apical cell layers in squamous epithelia, homogeneously expressed Ber-EP4 antigen. As Ber-EP4 does not detect any normal or neoplastic non-epithelial cells, this antibody might prove valuable for the differentiation of the following (i) non-epithelial tumours from undifferentiated carcinomas; (ii) hepatocytes from bile duct cells in certain liver diseases; (iii) mesothelial cells from carcinoma cells in lung biopsy specimens; and (iv) reactive mesothelial cells from carcinoma cells in smears of serous effusions.

Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations.

E-cadherin, a transmembrane cell adhesion molecule, has been observed to have an altered pattern of immunoreactivity in several types of carcinomas. In lobular breast cancer, loss of immunoreactivity has been shown to be due either to out-of-frame deletions or to nonsense mutations of the E-cadherin gene. We analysed 29 cases of completely resected colon carcinoma with immunohistochemistry using the HEC-D1 antibody. Normal protein expression similar to that in the adjacent nonmalignant mucosa was seen in 6 cases, whereas 23 tumours had reduced or absent E-cadherin expression. In the 8 cases with no expression of E-cadherin revealed by immunohistochemistry, the entire E-cadherin cDNA sequence was analysed. In these cases, sequence analysis failed to reveal any cDNA mutations despite the negative immunohistochemistry. Possible explanations for this discrepancy include regulatory defects in the E-cadherin promoter, abnormalities at the translation or protein processing levels and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g. intronic sequences), which could play a role in causing abnormal processing of the E-cadherin protein.

Triple malignancy of the genitourinary tract.

A patient is reported on who metachronously presented with renal cell carcinoma, urothelial cancer of the bladder and prostatic cancer.

Histopathological response after preoperative radiochemotherapy in rectal carcinoma is associated with improved overall survival.

BACKGROUND: Recent studies showed improved local control after preoperative radiochemotherapy (RCTX) in patients with locally advanced rectal carcinoma, but failed to demonstrate a survival benefit. Our aims were to determine outcome and impact of histopathological response after preoperative RCTX. METHODS: One hundred four patients with uT3 rectal carcinoma were treated with preoperative RCTX of 45 Gy and continuous 5-FU infusion between 1997 and 2001 (group I). Histopathological response was evaluated in all specimens after tumor resection. Group II consisted of 114 patients with uT3 rectal carcinoma treated with postoperative RCTX between 1988 and 1997. RESULTS: Group I showed a 6.1% 5-year local recurrence rate compared to 15.3% in group II (P = 0.023). Overall survival rates did not differ significantly between both groups (P = 0.225). Histopathological responders had a significantly improved 5-year overall survival with 89.1 (7.8)% compared to 68.7 (6.7)% of the non-responders (P = 0.008) and were identified as an independent prognostic factor. CONCLUSIONS: Significant improvement of overall survival was observed for histopathological tumor responders after neoadjuvant radiochemotherapy. Our protocol of preoperative radiochemotherapy confirms the results of the multi-center studies in regard to local control and overall survival.

Free peritoneal tumour cells are an independent prognostic factor in curatively resected stage IB gastric carcinoma.

BACKGROUND: Several studies have shown that the cytological detection of free peritoneal tumour cells (FPTCs) in patients with gastric cancer indicates the presence of metastatic disease. The immunocytochemical detection of FPTCs, especially in early-stage tumours, has not been examined comprehensively. METHOD: Peritoneal lavage was performed in 351 patients before curative resection of a gastric carcinoma between 1987 and 2001, and an adequate sample was obtained from 346 patients. FPTCs were detected immunocytochemically using Ber-EP4 antibody. Median follow-up time was 70 months. RESULTS: FPTCs were detected in the lavage fluid of 74 patients (21.4 per cent) and correlated with increasing pathological tumour depth (pT) and lymph node (pN) status (P < 0.001). The 5-year overall survival of patients with FPTCs was significantly worse than that of patients without FPTCs (35 versus 71.9 per cent; P < 0.001). FPTCs were present in 14 (8.5 per cent) of 164 patients with stage IA or IB tumours. Although the detection of FPTCs had no prognostic significance for stage IA tumours, the presence of FPTCs in those with stage IB tumours was associated with a worse prognosis (P < 0.001). Multivariate analysis identified the presence of FPTCs as an independent prognostic factor in the whole cohort and in the stage IB subgroup. CONCLUSION: Detection of FPTCs is associated with poor prognosis even in patients with early-stage gastric cancer and should be used for risk-group stratification.

Fatal relapse in Goodpasture's syndrome 3 years after plasma exchange.

Since 1919 about 400 cases with Goodpasture's syndrome have been published, and since 1975 about 100 of these patients were treated with plasma exchange in addition to immunosuppression. A remission was achieved in 50% of the 100 cases, and in 7 a relapse was reported. In this paper, we are reporting a case with a fatal relapse 3 years after successful plasma exchange treatment. This patient was a smoker, she had been exposed to hydrocarbon solvents, and she had the HLA tissue antigens DR2 and MT3. Lung biopsy by fiberoptic bronchoscopy revealed linear staining of IgG at the alveolar basement membrane. During relapse pulmonary infiltrations and macrohematuria did not respond a second time to methylprednisolone pulses nor to plasma exchange treatment and the patient died of massive lung hemorrhage.

[Platelet aggregation inhibition with calcium dobesilate]. / Thrombozytenaggregationshemmung unter Calciumdobesilat.

In 52 patients with pathologically increased platelet aggregation the effect of a 4-day oral treatment of 500 mg, 1000 mg and 1500 mg calcium dobesilate (Dexium 500) on spontaneous platelet aggregation was assessed using PAT III of Breddin. In comparison to placebo 1500 mg calcium dobesilate reduced platelet aggregation significantly, 1000 mg and 500 mg calcium dobesilate, however, didn't influence platelet aggregation.

[Minimal residual disease in gastrointestinal tumors: tumor detection in bone marrow, blood and lymph nodes]. / Minimale residuale Tumorerkrankung bei gastrointestinalen Tumoren: Tumorzellnachweis in Knochenmark, Blut und Lymphknoten.

The cure of a tumor patient with gastrointestinal cancer is dependent on the extension of the primary tumor (TNM-classification) and the option of curative resection (R0-resection) at the time of operation. The additional application of multimodal therapy approaches has lead to an improvement of prognosis in different advanced tumor stages. Nevertheless, despite curative tumor resection about 50% of patients with locally advanced gastrointestinal cancer develop recurrent tumor disease or distant metastases and die tumor-related. A possible explanation is the seed of disseminated tumor cells in blood, bone marrow or lymph nodes pre-, intra- or postoperatively, but also during diagnostic procedures. Several studies have shown in the last years that the presence of minimal residual disease (MRD) influences the course of disease and the patient's prognosis after curative tumor resection. Although several groups have reported the prognostic impact of disseminated tumor cells in the different compartments of bone marrow, lymph nodes and blood, the phenomenon of minimal residual disease is not acknowledged as an established prognostic factor and is not integrated into the classification of the UICC. Therefore, no therapeutic consequences were drawn at present from the detection of disseminated tumor cells in patients with gastrointestinal cancer. A possible explanation are missing multi-center-studies, which confirm the results of the several single-center-studies. Standardization of study designs and methodical procedures and the evidence of reproduction are mandatory in order to value and interpret the multitude of studies and the available data in this field. Only these results will allow to decide if the presence and detection of disseminated tumor cells can alter the tumor staging and individualize or possibly minimize further oncological therapy strategies.

[Polypoid tumors of the esophagus. Pseudosarcoma and carcinosarcoma]. / Polypoide Tumoren des Osophagus. Pseudosarkom und Karzinosarkom.

We report two cases of a carcinosarcoma and a pseudosarcoma of the esophagus and discuss the clinical, radiologic and histopathologic findings.

[Diffuse stomach cancer in the minor form of familial adenomatosis coli. Correlation or coincidence?]. / Diffuses Magenkarzinom bei der Minor-Form der familiären Adenomatosis coli. Zusammenhang oder Zufall?

We report about a 66-year-old female patient with Gardner syndrome, a special form of familial adenomatosis coli, and a stomach carcinoma occurring at the same time. Recently, there have been more reports about duodenal carcinomas and stomach polyps in intestinal adenomatosis due to an improvement in diagnostic methods. The concomitant occurrence of a stomach carcinoma is, however, a very rare finding. So far, altogether 10 case histories have been published world-wide. The case presented here and a review of the literature again lend current interest to the hypothesis formulated by Amman in 1976 according to which the occurrence of a stomach carcinoma in connection with a Gardner syndrome/familial adenomatosis coli, particular in the minor form of this entity (recessive hereditary course, less than 100 polyps in the intestines, occurrence of polyps in old age) must be regarded as an additional characteristic of this disease. On the basis of this hypothesis, all adenomatosis patients should undergo gastroscopy.

[Gastrointestinal disorders in psoriatic arthritis]. / Gastrointestinale Störungen bei Arthritis psoriatica.

Systemic amyloidosis is a rare complication in psoriatic arthritis, 16 cases only were published until now. Our case report is characteristic of gastrointestinal symptoms like chronic diarrhea, malabsorption, and intestinal pseudoobstruction. In addition, in this connection the first report of segmental pseudomembranous enteritis is given. Discussing the findings, observations and literature, the difference between psoriatic enteropathy, malabsorption in systemic amyloidosis and pseudomembranous enteritis in amyloidosis is shown. In this connection the basic different pathophysiological mechanisms are demonstrated.

Impact of cytokeratin-20 and carcinoembryonic antigen mRNA detection by RT-PCR in regional lymph nodes of patients with colorectal cancer.

The reported rates for tumour cell involvement in the locoregional lymph nodes of colorectal cancer vary greatly, depending on the method used and case selection. In order to further evaluate the clinical value of molecular biologic detection of tumour cells we investigated 102 histologically tumour-free (pN0) regional lymph nodes from 51 consecutive, completely resected (UICC R0) colorectal carcinoma specimens for the presence of tumour cell mRNA by RT-PCR specific for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20). Two lymph nodes located nearest to the primary tumour were investigated in each case. CK-20 mRNA was found in 31 of 51 patients (60.8%) and CEA mRNA in 30 of 51 patients (58.8%), respectively. Identical transcription patterns of CK-20 and CEA mRNA (both positive or both negative) were found in 38 of 51 patients (74.5%). There was a significantly higher proportion of cases with CEA positivity in the lymph nodes of tubulopapillary than of mucinous adenocarcinomas (P< 0.03). Detection of CK-20 and CEA mRNA correlated in nine of 12 cases (75.0%) with the risk of tumour recurrence (not significant) and showed a tendency towards shorter disease-free survival by univariate analysis (not significant). Our data indicate that CK-20 and CEA mRNA detection by RT-PCR may prove useful for the prediction of tumour recurrence of patients with pN0 colorectal carcinoma, although neither reach statistical significance in this series of patients.