Rainer Broll

VEGF isoforms and mutations in human colorectal cancer

We wished to demonstrate vascular endothelial growth factor (VEGF) transcript polymorphism in human colon cancer. RNA was extracted from 25 primary human colorectal adenocarcinomas followed by VEGF transcript amplification, fragment elution, subcloning, positive selection via insert analysis and sequencing. Four distinct splice variants were consistently expressed in cancer, including VEGF121, VEGF165, VEGF189 and the newly identified truncated splice variant VEGF145. Six novel mutations were characterized, all of which occurred within the conserved expression site of the gene and which consequently were present in all splice forms. Five cancers exhibited single nucleotide changes and 1 cancer a 2‐nucleotide deletion. A silent mutation was observed in exon 1 at position +70 relative to the amplification start site, a 1‐ and 2‐base deletion with frameshift and protein truncation in exon 3 at positions +172 and +171/172, respectively, a transition mutation in exon 3 at position +248 and 2 transition mutations in exon 4 at positions +398 and +403. All of these sense mutations should alter protein conformation. To our knowledge, this is the first report of VEGF145 in solid malignancy. Its biologic activity remains to be determined. We have demonstrated a variety of sporadic mutations within human colorectal cancer VEGF mRNA. Mutant angiogenic VEGF may provide a genomic basis for the diversity of tumor‐host response and may prove to be important in antisense oligonucleotide targeting, since all the different VEGF isoforms would have to be neutralized to prevent angiogenesis.

p21, p27, cyclin D1, and p53 in rectal cancer: immunohistology with prognostic significance?

Abstract.Background and aims: This study examined the prognostic value of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1, and the cell cycle regulating proteins cyclin D1 and p53 after curative surgery for rectal cancer. Patients and methods: Formalin-fixed, paraffin-embedded tissue samples of 160 rectal carcinomas resected curatively within a 5-year period were used. Immunohistochemical analysis used monoclonal antibodies p21Waf1/Cip1 (clone SX118), p27Kip1 (clone SX53G8), cyclin D1 (clone DCS-6), and p53 (DO-1). Positive nuclear protein expression was assessed at the 10% level. Results of immunohistochemistry were studied for correlation with clinical and histopathological data of the prospective tumor registry including recurrence and patient survival. Results: Of the 160 rectal carcinomas 36% were p21Waf1/Cip1 positive, 44% p27Kip1 positive, 48% cyclin D1 positive, and 39% p53 positive. The p21Waf1/Cip1 staining pattern was correlated with p27Kip1 and p53 expression and with UICC stage and lymph node status. p53 status was not correlated to any clinical or histopathological variable. p27Kip1 expression was associated with tumor size and cyclin D1 expression. Tumor progression caused by local and distant recurrence occurred in 20%. p21Waf1/Cip1, p27Kip1, and p53 were strong predictors of recurrence. p21Waf1/Cip1 and p53 but not p27Kip1 were independently correlated with disease-free survival. UICC stage was independently related to both recurrence and survival. The best prognosis was in p21Waf1/Cip1 positive and p53 negative rectal carcinomas. Conclusions: Reflecting tumor biology by immunohistochemical assessment of cell cycle regulators, p21Waf1/Cip1 and p53 were independently predictive of prognosis in rectal cancer, and p27Kip1 was independently related to recurrence. However, cyclin D1 had no independent relationship to prognosis. Clinically, UICC stage was a strong predictor of prognosis after curative surgery for rectal cancer.

The proliferation marker pKi‐67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B

The proliferation marker pKi‐67 (‘Ki‐67 antigen’) is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell‐cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi‐67, using a yeast two‐hybrid system. A mammalian two‐hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell‐cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA‐structural and nucleolus‐associated proteins binding to pKi‐67 were found. Moreover, it was demonstrated that the N‐terminal domain of pKi‐67 is capable of self‐binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi‐67 on the localization of cell‐cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline‐responsive gene expression system was used to induce the pKi‐67 fragments previously used for the two‐hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi‐67 is a Ran‐associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M‐phase. Copyright

Prognostic significance of free gastrointestinal tumor cells in peritoneal lavage detected by immunocytochemistry and polymerase chain reaction

Abstract. Aims: The aim of our study was to identify tumor cells in peritoneal lavage comparatively with immunocytochemistry (ICC) and half-nested reverse transcriptase-polymerase chain reaction (RT-PCR) using carcinoembryonic antigen (CEA) as marker and to evaluate their prognostic significance. Patients and methods: In 75 patients who underwent surgery for a carcinoma of the colorectum (n=49), stomach (n=17) or pancreas (n=9) and 13 patients with an abdominal aortic aneurysm (control group) the abdomen was irrigated with saline solution immediately after laparotomy. Cells were separated by Ficoll-density centrifugation and divided into 2 equal volumes for ICC and RT-PCR. For ICC cells were spun onto slides by cytospin centrifugation and stained with a monoclonal antibody (mab) against CEA using the APAAP method. For RT-PCR total RNA was extracted from the cells, transcribed into cDNA and amplified with CEA-specific primers. Lavages of 13 patients with an abdominal aortic aneurysm and blood samples of 6 healthy donors served as controls. Results: Immunostained tumor cells were found in peritoneal lavage in 23% (17/75) of all patients, whereas 63% (47/75) of patients gave a positive result by RT-PCR analysis. In the control group (n=13) no patient presented with tumor cells in ICC, however 5 of 13 (38%) showed amplified CEA-mRNA by RT-PCR, and so did one of six blood samples. Using ICC technique, we found significant correlations between detection rates and pT-, pN-, pM-categories as well as tumor stage. On the contrary, by RT-PCR significant correlations were observed only between pT- and pM-categories and detection rates. Detection of tumor cells in peritoneal lavage with both techniques was associated with poor prognosis. Moreover, these tumor cells are an independent prognostic factor and may have an influence on the development of peritoneal carcinomatosis. Conclusion: ICC is a useful method for detection of tumor cells in peritoneal lavage. In contrast, half-nested RT-PCR cannot be recommended, as the detection rates are unproportionally high, obviously as a result of CEA-mRNA expression in nontumor cells.

Tumorzelldissemination in das Knochenmark und in die Peritonealhohle eine immunzytochemische Untersuchung an Patienten mit einem Magen- oder kolorektalen Karzinom

The tumor spread and the radicality of surgical resection are the most important facts in a patients prognosis. In spite of curative tumor resection many patients die from metastases or local tumor recurrence. One possible reason is early dissemination of tumor cells which cannot be detected with clinical methods of examination. For this reason the aim of our study was to examine both bone marrow and peritoneal lavage for disseminated tumor cells with an immunocytochemical technique in patients with a gastrointestinal carcinoma. We also wanted to find out whether there was any correlation between the incidence of tumor cell detection and the TNM classification, staging and tumor grading and whether disseminated tumor cells have any prognostic significance. Our study included 54 patients who underwent surgery in our clinic for a carcinoma of the stomach (20 patients) or the colorectum (34 patients) from November 1993 to December 1994. At the beginning of the operation bone marrow had been taken from the iliac spine, and the abdomen was irrigated with 1000 ml saline solution immediately after laparotomy or laparoscopy. After cell separation with Ficoll density centrifugation 5 × 105 cells were applied per slide by a cytospin technique. For detection of the tumor cells we used the APAAP technique and the following monoclonal antibodies: KL1, CK2, anti-CEA, 17-1A (bone marrow) and Ber-EP4, B72.3, anti-CEA and 17-1A (peritoneal lavage). Altogether 77% of all patients had tumor cells in the bone marrow and 69% in peritoneal lavage fluid. It was possible to detect tumor cells in bone marrow (67%) and peritoneal lavage fluid (25%) even of patients with T1 tumors. The percentage increased with depth of wall infiltration. There was a marked difference in bone marrow aspirates between patients with lymph-node-negative tumors (N0) and those with lymph-node-positive tumors (N+): 65% had tumor cells in N0 and 85% in N+ stages. This trend was also seen in patients with (M1) and without (M0) metastases, in both bone marrow aspirates and peritoneal lavage fluid. In bone marrow there was a good correlation of tumor cells with staging, but in peritoneal lavage fluid this was not so. Finally, we detected tumor cells more often in bone marrow and peritoneal lavage fluid of patients with poorly differentiated tumors (G3) or diffuse Laurén type than in patients with moderately differentiated tumors (G2) or intestinal Laurén type. After a median follow-up period of 12.5 months patients with disseminated tumor cells had a lower survival rate than patients without tumor cells.ZusammenfassungDie Tumorausdehnung und die Radikalität des chirurgischen Eingriffs sind für die Prognose eines Patienten von großer Bedeutung. Trotz kurativer Resektion des Tumors versterben jedoch viele Patienten an den Folgen der Fernmetastasierung oder einem lokalen Rezidiv. Die Ursache dafür könnte eine frühe Dissemination von Tumorzellen sein, welche derzeit mit den herkömmlichen klinischen Untersuchungsmethoden noch nicht erfaßt werden kann. Aus diesem Grunde war es das Ziel unserer Studie, das Knochenmark und die Peritonealspülflüssigkeit von Patienten mit einem gastrointestinalen Karzinom auf Tumorzellen mit immunzytochemischer Technik zu untersuchen. Außerdem wollten wir wissen, ob eine Korrelation zur TNM-Klassifikation, zum Tumorstadium und zum Tumordifferenzierungsgrad vorliegt und ob dem Tumorzellnachweis prognostische Relevanz zukommt. WirThe tumor spread and the radicality of surgical resection are the most important facts in a patients prognosis. In spite of curative tumor resection many patients die from metastases or local tumor recurrence. One possible reason is early dissemination of tumor cells which cannot be detected with clinical methods of examination. For this reason the aim of our study was to examine both bone marrow and peritoneal lavage for disseminated tumor cells with an immunocytochemical technique in patients with a gastrointestinal carcinoma. We also wanted to find out whether there was any correlation between the incidence of tumor cell detection and the TNM classification, staging and tumor grading and whether disseminated tumor cells have any prognostic significance. Our study included 54 patients who underwent surgery in our clinic for a carcinoma of the stomach (20 patients) or the colorectum (34 patients) from November 1993 to December 1994. At the beginning of the operation bone marrow had been taken from the iliac spine, and the abdomen was irrigated with 1000 ml saline solution immediately after laparotomy or laparoscopy. After cell separation with Ficoll density centrifugation 5 x 10(5) cells were applied per slide by a cytospin technique. For detection of the tumor cells we used the APAAP technique and the following monoclonal antibodies: KL1, CK2, anti-CEA, 17-1A (bone marrow) and Ber-EP4, B72.3, anti-CEA and 17-1A (peritoneal lavage). Altogether 77% of all patients had tumor cells in the bone marrow and 69% in peritoneal lavage fluid. It was possible to detect tumor cells in bone marrow (67%) and peritoneal lavage fluid (25%) even of patients with T1 tumors. The percentage increased with depth of wall infiltration. There was a marked difference in bone marrow aspirates between patients with lymph-node-negative tumors (N0) and those with lymph-node-positive tumors (N+): 65% had tumor cells in N0 and 85% in N+ stages. This trend was also seen in patients with (M1) and without (M0) metastases, in both bone marrow aspirates and peritoneal lavage fluid. In bone marrow there was a good correlation of tumor cells with staging, but in peritoneal lavage fluid this was not so. Finally, we detected tumor cells more often in bone marrow and peritoneal lavage fluid of patients with poorly differentiated tumors (G3) or diffuse Lauren type than in patients with moderately differentiated tumors (G2) or intestinal Lauren type. After a median follow-up period of 12.5 months patients with disseminated tumor cells had a lower survival rate than patients without tumor cells.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma

PurposeThe present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients.MethodsImmunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy.ResultsWe determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3–66.4%), and a mean mRNA level of 0.1769 (ΔCT: range 0.01–0.69); indices and levels did not correlate. High pKi-67 mRNA ΔCT values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors.ConclusionQuantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Proliferation marker pKi‐67 affects the cell cycle in a self‐regulated manner

The proliferation marker pKi‐67 is commonly used in research and pathology to detect proliferating cells. In a previous work, we found the protein to be associated with regulators of the cell cycle, controlling S‐phase progression, as well as entry into and exit from mitosis. Here we investigate whether pKi‐67 has a regulative effect on the cell cycle itself. For that purpose we cloned four fragments of pKi‐67, together representing nearly the whole protein, and an N‐terminal pKi‐67 antisense oligonucleotide into a tetracycline inducible gene expression system. The sense fragments were C‐terminally modified by addition of either a nuclear localization sequence (NLS) or a STOP codon to address the impact of their intracellular distribution. FACS based cell cycle analysis revealed that expression of nearly all pKi‐67 domains and the antisense oligonucleotide led to a decreased amount of cells in S‐phase and an increased number of cells in G2/M‐ and G1‐phase. Subsequent analysis of the endogenous pKi‐67 mRNA and protein levels revealed that the constructs with the most significant impact on the cell cycle were able to silence pKi‐67 transcription as well. We conclude from the data that pKi‐67 influences progression of S‐phase and mitosis in a self‐regulated manner and, therefore, effects the cell cycle checkpoints within both phases. Furthermore, we found pKi‐67 mediates an anti‐apoptotic effect on the cell and we verified that this marker, although it is a potential ribosomal catalyst, is not expressed in differentiated tissues with a high transcriptional activity. J. Cell. Biochem. 87: 334–341, 2002.

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects

The Ki‐67 antigen, pKi‐67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi‐67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi‐67 N‐terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi‐67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi‐67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi‐67 N‐terminus is differentially spliced resulting in at least five different isoforms with different functions.

Assessment of the proliferation index in gastric carcinomas with the monoclonal antibody MIB 1

Abstract Our study aimed to reveal whether the proliferation index of tumor cells, calculated with the monoclonal antibody (mAb) MIB1, is of prognostic relevance in patients with a gastric carcinoma and shows any correlation to well-known clinicopathological factors (TNM categories, stage, grade, Laurén type). We examined formalin-fixed, paraffin-embedded tissue blocks of samples from 94 patients, who underwent surgery for an adenocarcinoma of the stomach between 1988 and 1991. Specimens were immunohistochemically stained using the mAb MIB1 in combination with the alkaline-phosphatase/anti-(alkaline phosphatase) technique. The proliferation index (PI) was estimated in various areas of interest (tumor center and periphery and in lymph node metastases of compartments I and II), by always counting 200 tumor cells in three different high-power fields per specimen, and calculated as the percentage of MIB1-positive tumor cell nuclei relative to all tumor cell nuclei in the area examined. The total PI in the primary tumor was 47.2% and slightly higher in the center (49.1%) compared to the periphery (44.7%). Surprisingly in lymph node metastases the PI was lower than in the primary tumor (compartment I: 39.5%, compartment II: 33.6%). Tumors with distant metastases revealed a higher proliferative activity (55.1%) than tumors without (44.3%). The PI increased significantly from well to poorly differentiated carcinomas (P < 0.01), whereas the intestinal Laurén type showed a lower PI than the diffuse type. No difference in survival was found between patients with a median PI or less and those with a PI above the median (47.2%). Our results show that the proliferation index in gastric carcinomas has no prognostic relevance and therefore is of low clinical value.

Metronomic trofosfamide inhibits progression of human lung cancer xenografts by exerting anti-angiogenic effects

BackgroundRecent studies of conventional chemotherapeutic drugs administered in metronomic therapy schedules showed remarkable inhibitory effects on tumor angiogenesis. Subsequent and prolonged tumor regression was achieved moreover by circumventing acquired drug resistance. In this study, metronomic and conventional trofosfamide were compared on human NSCLC xenograft “LX-1.”Materials and methodsIn vitro cytotoxicity of trofosfamide on tumor and human umbilical cord endothelial cells was determined under normoxic and hypoxic conditions. Additionally fractions and duration of cell cycles were analyzed by flow cytometry. In vivo LX-1 xenotransplanted nude mice were treated with trofosfamide in conventional and metronomic schedules (i.p./p.o.). Tumor sections were evaluated for microvessel density (MVD), relative growth fraction and apoptosis.ResultsIn contrast to the rapid growth of conventionally treated lung cancer, long lasting tumor growth retardation over the total treatment period was achieved with metronomic treatment. While growth fraction and apoptotic rate of LX-1 cells remained unchanged, the MVD was significantly reduced (50%).ConclusionOur results show advantages of a metronomic trofosfamide schedule compared to a conventional bolus therapy mainly due to inhibition of angiogenesis. In vitro data show that this mechanism works under normoxic and hypoxic conditions and suggest that this is in part a direct cytotoxic effect on endothelial cells.