Search

Background: The direct comparison of CA19.9, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has never been performed for the diagnosis of solid pancreatic tumours (SPTs)., Methods: We included 68 patients with a SPT referred for EUS-FNA. CTCs were analysed using size-based platform and ctDNA using digital PCR. The sensitivity, specificity, negative and positive predictive values were evaluated for each marker and their combination., Results: SPTs corresponded to 58 malignant tumours (52 pancreatic adenocarcinoma (PA) and 6 others) and 10 benign lesions. The sensitivity and specificity for PA diagnosis were 73% and 88% for EUS-FNA, 67% and 80% for CTC, 65% and 75% for ctDNA and 79% and 93% for CA19.9, respectively. The positivity of at least 2 markers was associated with a sensitivity and specificity of 78% and 91%, respectively. CtDNA was the only marker associated with overall survival (median 5.2 months for ctDNA+ vs 11.0 months for ctDNA-, P=0.01)., Conclusions: CA19.9 alone and in combination with ctDNA and/or CTC analysis may represent an efficient method for diagnosing PA in patients with SPTs. Further studies including a larger cohort of patients with both malignant and benign lesions will be necessary to confirm these promising results.

Biallelic and heterozygous mutations of the BUB1B gene have been reported in mosaic variegated aneuploidy (MVA), a rare disorder characterized by constitutional mosaic aneuploidies associated to severe intrauterine growth retardation, microcephaly and, in most cases, to premature chromatid separation (PCS), highlighting the key role of human BUBR1 in chromosome segregation. To study the consequences of gradual reduction of the BUBR1 protein levels, inhibition of BUB1B expression in model cells was induced using short hairpin RNAs (shRNAs). We obtained stable shRNA-transduced HeLa cells displaying a gradient of residual BUBR1 protein (8.5, 10, 14, 58, and 77%), mimicking the situation of patients' cells harboring one or two BUB1B mutations. Induction of PCS was detected in all transduced cells and its level was correlated to the decrease of BUBR1. Aneuploidy was clearly detected in cells with residual BUBR1 below 50%. Our data demonstrate that the function of the human BUBR1 protein in the spindle checkpoint is remarkably dosage-dependent and that the biological consequences of BUB1B expression reduction on premature chromatid separation and aneuploidy depend on the residual amount of BUBR1. This provides a biological explanation for the mode of inheritance of PCS, which is dominant, and of MVA, which can be recessive in some families and result from the combination of a null allele associated to a common hypomorphic allele in others.

We compared three different means of assaying tumor proliferative activity in 30 human colorectal adenocarcinomas labeled in vivo with bromodeoxyuridine (BrdUrd). The labeling indices (LI) of BrdUrd obtained both by flow cytometry (FCM) and immunohistochemistry (IH) were also compared with the labeling index of Ki-67. These methods were then related to tumor ploidy and pathological features. Flow cytometry was performed in accordance with Begg's method after intravenous infusion of BrdUrd four hours before surgery. Immunohistology was carried out on paraffin-embedded sections with monoclonal antibodies against BrdUrd and Ki-67. A positive correlation was found between BrdUrd LI obtained by both FMC and IH (p<0.0001), a finding that complies with the literature. However, we report on a correlation between Ki-67 LI and BrdUrd LIs in colorectal tumors (p=0.012). The results were valid for all tumors when they were subdivided into diploid and aneuploid groups. The labeling indices were significantly higher in the aneuploid tumor group than in the diploid group (p=0.047). No relationship between proliferation parameters and tumor stage or grade was found. To our knowledge, this is the first report on a positive correlation between tumor proliferation indices in BrdUrd LIs and Ki-67 in colorectal carcinomas. This finding validates the value of Ki-67 immunostaining, which, however, should be confirmed in a larger series under the same technical conditions.

In colorectal cancer, tumoral kinetic parameters could influence the therapeutic strategy. The aim of this study was to test the validity of a preoperative assessment of tumoral kinetics on biopsies obtained from endoscopic sampling. After infusion of 250 mg of bromodeoxyuridine (BrdUrd), tumor samples were taken. The following kinetic parameters were evaluated by Begg's method: labeling index (LI) of BrdUrd, duration of the S phase (Ts), and potential doubling time (Tpot). We compared the findings in preoperative endoscopic samples (t1), endoscopic samples (t2), and macrobiopsies of the tumor after surgical resection (t3) to test the effect of time, surgical procedures, and sampling mode on results. Eight diploid and 11 aneuploid colorectal cancers were studied. In diploid tumors, no difference in Tpot, Ts, or LI values was found among the three time intervals. In aneuploid tumors, Tpot value at t1 was lower than t3: 2.9 +/- 1.2 versus 4.5 +/- 1.4 days (P = 0.02). This difference seemed to be independent of sampling type but related instead to the time interval between BrdUrd infusion and biopsy and to the changes due to the surgical condition. On the other hand, LI seemed to be the most stable kinetic parameter. With a cutoff value of 4 days for Tpot and of 10% for LI to differentiate slow and fast growing tumors, the positive predictive values of LI were 90% at t1, 80% at t2, and 50% at t3 in aneuploid tumors.

Somatostatin receptor are transiently expressed by immature granule cells of rat cerebellum. The effects of somatostatin and octreotide on cell proliferation were studied in cultured cerebellar explants from 10-day-old rats. Cell multiplication was measured using [3H]thymidine incorporation and flow cytometric analysis of cell cycle parameters. [3H]Thymidine incorporation occurred exclusively in neuroblasts of the external granule cell layer in the presence of insulin (1 microM). The labeling index, the length of the S phase, and the potential doubling time were similar in vivo and in explants. Octreotide (10(-12) to 10(-6)M) had no effect on [3H]thymidine incorporation in cerebellar explants. In addition, somatostatin and octreotide did not modify the proportion of cells in the S, G0-G1, and G2-M phases. The present results demonstrate that somatostatin does not affect cell multiplication in neurons of the external granule cell layer.

Mitotically active Schwann cells isolated from adult rat sciatic nerve segments were cultivated, using a bivariate BrdU/DNA flow cytometry analysis, to test the effect of b-FGF (10 ng/ml), alpha-MSH (10 ng/ml), NGF (10 ng/ml), PDGF-AB (10 ng/ml), and TGF-beta 1 (10 ng/ml) on the cell cycle. Compared to control or cholera toxin-treated cultures, no significant differences (P < 0.05; Newmann-Keuls test) were observed in the proportion of G0G1 cells, S cells, G2M cells, and in the LI when alpha-MSH, NGF, PDGF-AB, or TGF-beta 1 were present. The S phase duration varied from 6.16 +/- 0.24 to 7.86 +/- 2.6 h, and the deduced potential doubling time was estimated at between 46.70 +/- 7.09 and 56.05 +/- 7.55 h. In contrast, when b-FGF was added to the culture, the cell cycle was significantly modified, and the proportion of G0G1 cells decreased from 68-77% to 59-64%, while the proportion of S cells increased from 14-16% to 24.0-26.4%. Although S phase duration was not significantly changed (6.02 +/- 0.36 h), the 1.7- to 2.8-fold LI increase reduced the potential doubling time to 25.99 +/- 6.13 h. We conclude from these results that only b-FGF-induced adult rat Schwann cells dramatically reenter in cell cycle and that this growth factor may be an axonally derived signal-promoting mitogenesis after nerve injury.

Parallel investigations of ploidy by flow cytometry and cytogenetics were performed in 20 colorectal tumors. Flow cytometry detected an aneuploidy in 13 tumors with DNA indices ranging from 1.13 to 2.21. The other samples exhibited an apparent diploid DNA content. Cytogenetic analyses revealed an abnormal chromosome count in 14 cases and a frequent implication of numerical or structural changes in chromosomes 1, 7, 12, 17, 18, and 20. DNA content evaluated by both techniques was generally concordant with minor discrepancies not exceeding 10%. In six cases, cytogenetics failed to find the cell populations detected by flow cytometry. These results indicate that flow cytometry and cytogenetics are reliable and complementary techniques, particularly in near-diploid tumors, where flow cytometry has some difficulties in detecting variations from diploidy below to 8%.

Chloroethylnitrosoureas are reactive compounds that are highly effective against malignant neoplasms in humans and animals. The most widely used nitrosoureas, lomustine and carmustine, are known to be hepatotoxic and to induce pericholangitis and intrahepatic cholestasis, which in the long term lead to cholangiolysis and biliary cirrhosis. However, the nitrosourea fotemustine has proved to be non-hepatotoxic at 20 mg/kg and 50 mg/kg. We have studied the effect of these three nitrosoureas on the cytotoxicity and cellular kinetics of rat liver cells. Lomustine and carmustine modify the proliferation index of liver cells in vivo: flow cytofluorometry showed that DNA cell distribution is quite similar for lomustine and carmustine, with subsequent accumulation of cells in G2 + M phase. 3 months later regressive morphological and cell cycle perturbations are noted for the lower dose of lomustine and carmustine. The most severe lesions are noted with lomustine (50 mg/kg). Fotemustine is not hepatotoxic and preferentially induces S phase perturbations. The more toxic nitrosoureas, lomustine and carmustine, induce comparable hepatocyte cell cycle alterations which differ from those induced by the less hepatotoxic nitrosourea fotemustine.