Ki-67 and c-jun expression in pancreatic cancer: a prognostic marker?

The proto-oncogene c-jun is involved in cell proliferation and Ki-67 antigen permits determination of the proportion of proliferating tumour cells. The expression of c-jun and Ki-67 in pancreatic cancer and their relation with tumour histological features and patients survival were evaluated. Specimens were obtained as follows: 14 pancreatic cancer from patients radically operated, 8 liver metastases from subjects submitted to palliation, 5 normal pancreas from organs donors and 5 chronic pancreatitis. Ki-67 and c-jun were studied by immunohistochemistry. The percentage of tumour cells stained for c-jun was increased in 11/14 cases. A high c-jun expression was more frequently found in liver metastases than in pancreatic cancer tissue (p=0.031). The frequency of high c-jun expression was more elevated in short-term as compared to long-term survivors (Fisher's exact test, p=0.031 and log-rank, p=0.03). The percentage of tumour positive cells for Ki-67 showed a mean value of 12.8% in primary pancreatic cancer and was lower than in the liver metastases (32.5%) (p=0.029). Significantly lower values were found in long-term (6.5%) as compared to the short-term survivors (18.1%) (p=0.032 and log-rank, p=0.006). A positive relation was demonstrated with stage (p<0.05), lymph node state (p=0.045) and perineural invasion (p=0.0001). In the multivariate analysis the Ki-67 staining was the most important determinant of long-term survival (p=0.005). c-jun and Ki-67 are overexpressed in pancreatic carcinoma, but only Ki-67 is a strong predictive factor.

Detection of B-cell monoclonality in fine needle aspiration by PCR analysis.

Cytologic examination of fine-needle aspiration (FNA) sometimes fails to diagnose the malignant nature of B-cell proliferations. In this study we analyzed the Ig gene rearrangement of 49 FNA samples by polymerase chain reaction (PCR) in order to evaluate whether molecular analyses can improve the accuracy of FNA. Twenty-six patients had non-Hodgkin's lymphoma, 11 had reactive lymphoid diseases, 5 had chronic inflammation and 7 had carcinoma. A semi-nested PCR was performed using an oligoprimer specific for consensus sequences of the V regions (FR3A) and two oligoprimers derived from conserved sequences of the J regions (LJH and VLJH). Histologic examination always followed the molecular and cytologic analysis. The sensitivity of PCR and FNA morphological examination in detecting a neoplastic pattern was 92% and 78%, respectively. When samples were considered inadequate for cytologic examination, PCR always reached a diagnosis consistent with the histologic features. Our results demonstrate that PCR analysis of FNA specimens is a reliable and sensitive method capable of enhancing the diagnostic accuracy of cytologic examination.

Heparin Induces Apoptosis in Lymphocytes from B-cell Chronic Lymphocytic Leukemia.

It has been shown that glycosaminoglycans play a role in the regulation of immune response. In particular, heparin exerts an antiproliferative and apoptotic action in different cellular systems. In this study we evaluate whether heparin can also induce a naturally occurring programmed cell death in lymphocytes from B-chronic lymphocytic leukemia (B-CLL), a neoplastic lineage where apoptosis is blocked by the expression of the proto-oncogene bc1-2. Peripheral blood lymphocytes (PBL) from 7 cases of B-CLL patients in different stages were cultured with three different heparin sodium concentrations for 4 days. Apoptosis was evaluated by agarose gel electrophoresis and by cytofluorimetric analysis. Bcl-2 expression was tested by flow cytometric analysis and immunohistochemistry on cytospin preparations. Agarose gel electrophoresis showed the characteristic DNA fragmentation pattern of apoptosis in all the cases of B-CLL stage III and IV after heparin incubation. DNA from normal and neoplastic lymphocytes cultured without heparin did not undergo spontaneous apoptosis. Cytofluorimetric analysis confirmed the agarose gel pattern and found a level of apoptosis over 50% after culture of neoplastic PBL with heparin. In these cases bcl-2 expression was found to be significantly reduced after heparin incubation when comparing to bcl-2 level before incubation. Our data adds further evidence regarding the potential role of heparin in oncogene inhibition and in apoptosis induction. In particular, the induction of apoptosis in neoplastic lymphocytes by heparin may have a role in the complicated field of interactions between the immune system and the blood vessels by glycosaminoglycans.

CD25-negative hairy cell leukaemia: intracytoplasmic detection of Tac antigen and interferon-induced surface expression.

Hairy cell leukaemia (HCL) is a chronic lymphoproliferative disease of B-cell lineage. One of the peculiar immunophenotypic markers is the strong expression of the p55 chain of the interleukin-2 receptor (IL2R), recognized by anti-CD25 (or anti-Tac) monoclonal antibody. However, it is known that in rare cases CD25 may not be detectable, even when variant forms of HCL are excluded. The possibility has not been investigated that in these situations CD25 is present in the cytoplasm of the neoplastic cells. This paper describes a case in which the clinical, histological, and electron microscopic features were consistent with a typical HCL. Immunophenotype analysis showed the whole spectrum of markers of HCL, except for the expression of IL2R. The soluble form of the molecule was, however, increased in the patient's serum. Cytospin staining of the neoplastic B cells with anti-CD25 clearly demonstrated the presence of IL2R in the cytoplasm of hairy cells. When the cells were cultivated in vitro in the presence of interferon-alpha 2b, CD25 was detectable at the membrane level. These findings suggest that at least some cases of CD25-negative HCL may express cytoplasmic IL2R.

Simultaneous visualization of immunodetected antigens and tissue components revealed by non-enzymatic histochemical stains.

We explored the possibility of simultaneous application of histochemical and immunohistochemical staining techniques on the same paraffin-embedded human tissue section. Conventional histological stains (PAS, Alcian, Alcian-PAS, Van Gieson, Gomori silver impregnation, and Giemsa) were used in association with a battery of markers (keratins, leucocyte common antigen, S-100 protein, Factor VIII-related antigen) that are widely employed in diagnostic and experimental studies. We found that the best procedure was to perform immunostaining before the histochemical reaction, as this enables all the other possible combinations to be carried out. In addition, several detection systems, such as peroxidase-anti-peroxidase (PAP), alkaline phosphatase-anti-alkaline phosphatase (APAAP), and avidin-biotin complex (ABC), were tested and all gave consistent results. Some minor modifications of the histological staining methods were necessary, but the current immunohistochemical techniques could be used as established. Preliminary findings indicate that immunohistochemistry can be combined with histochemistry techniques by means of a relatively simple procedure whose only disadvantage is the time required to carry out the double staining.