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Background. In gastric cancer (GC) the expression of HER2 is known as a marker of prognosis and recently it has been confirmed as a predictive marker of response to trastuzumab. Methods. GC specimens of 42 patients were collected. Representative samples from both primary tumors (42 samples) and lymph node metastases (23 samples), were selected. In each case, 4B5 (Ventana), CB11 (kit Oracle Menarini), HercepTest (Dako) antibodies were tested in immunohistochemistry (IHC) and scored as proposed for GC. HER2 gene status was studied by double probe fluorescence in situ hybridization (FISH) in all cases. Concordance among IHC scoring results of the 3 antibodies and between FISH results and IHC (0/1+ and 2+/3+), independently from the percentage of positive cells, were evaluated using the Cohen-Fleiss’ kappa statistic (K). Then, the number of specimens needed to be tested in cases with < 10% of HER2 overexpression was assessed. Finally, influence of gain of CEP17 (copies number > 3) on the results of FISH ratio was considered. Results. The 3 antibodies showed a K of 0,76 (p < 0,05). In the primary tumor, the overall concordance of FISH/IHC, taking into account the results of at least one antibody, was of 0,95 (p < 0,05). FISH/IHC concordance decreased to 0,82 (p < 0,05) when correlated with lymph node metastases. Five cases showed 2+/3+ score values in < 10% of cells. In 4 of these cases the percentage increased to > 10% adding 2 more sections from different tissue blocks of the primary tumor. In our results, the gain of CEP17 did not influence the final score ratio of FISH analysis. In conclusions, the HER2 analysis in GC needs a specific protocol avoiding working over-load and to solve equivocal cases.

Background: In gastric cancer (GC) the expression of HER2 is known as a marker of prognosis and recently it has been confirmed as a predictive marker of response to Trastuzumab. Design: GC specimens of 100 patients were collected. Representative samples from both primary tumors (100 samples) and lymph node metastases (24 samples), were selected. In each case, 4B5 (Ventana), CB11 (kit Oracle Menarini), HercepTest (Dako) antibodies were tested in immunohistochemistry (IHC) and scored as proposed. HER2 gene status was studied by double probe fluorescence in situ hybridization (FISH) in all cases. Concordance among IHC scoring results of the 3 antibodies and between FISH results and IHC (0/1+ and 2+/3+), independently from the percentage of positive cells, were evaluated using the Cohen-Fleiss’ kappa statistic (K). The number of specimens needed to be tested in cases with 3) on the results of FISH ratio was considered. Results: The 3 antibodies showed a K of 84% (p80% (p85% (p10% adding 2 more sections from different tissue blocks of the primary tumor. In our case series, the gain of CEP17 did not influence the final score ratio of FISH analysis. Conclusions: The HER2 analysis of surgical specimens of GC has to consider the tumor heterogeneity. When the IHC score is 0/1+ on 1 tissue block, we recommend to test 2 more tissue blocks, particularly in the cases were the negative score is related to the low percentage of positive cells (

The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

To the Editor: Ricci et al1 reported that human peripheral blood lymphocytes express α1-adrenergic receptor subtypes. The authors present results of radioligand binding assays with [3H]-prazosin as the radioligand for α1-adrenergic receptors, showing that the maximal density of the binding sites is 175 fmol per 106 cells. This would mean that the actual number of receptors per cell would be exactly 100 000, which is an unusually high number of receptors. Cell lines that are known for their high expression of α1-adrenergic receptors have been reported to express 10 000 to 25 000 receptors per cell.2 3 We have to keep in mind, however, that these numbers are found on kidney or smooth muscle cells, which are much larger than the freshly isolated lymphocytes that were used in the study by Ricci. Moreover, it is known for other G-protein–coupled receptors, eg, β2-adrenergic receptors, that are expressed on lymphocytes that the actual number of receptors per cell is around 2000.4 Thus, the number reported by Ricci et al for α1-adrenergic receptors on lymphocytes is extraordinary high. The authors do cite the literature on α1-adrenergic receptor expression in peripheral blood lymphocytes only partly correct. The only study that reports α1-adrenergic receptor binding on lymphoid cells from peripheral blood is the study by Jetschmann et al.5 However, these authors only examined NK cells where they reported the presence of 600 to 700 receptors per cell. Casale at al6 could not demonstrate binding sites on peripheral blood lymphocytes, whereas Maestroni et al7 only investigated α1-receptor expression …

Different normal and malignant human B-cell populations were studied with a twofold aim: to define which cytokines are produced in vivo, and to assess the relationship between cytokine production and kinetic state. To analyse normal B-cells representative of different stages of activation and proliferation in vivo, we purified germinal centre (GC)-B blasts and mantle B (M-B) cells from tonsils. To compare malignant B lymphocytes with their closest normal equivalent cells, we separated malignant CD5+B lymphocytes from the peripheral blood of patients with B-chronic lymphocytic leukemia (B-CLL) and normal CD5+B lymphocytes from cord blood. The expression of interleukins (IL) IL-1 alpha, IL-1 beta, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), IL-2, IL-4, and IL-6 genes was analysed using Northern and Western blotting techniques. TNF-alpha mRNA is produced by resting (M-B) and actively proliferating (GC-B) normal B lymphocytes. TGF-beta mRNA is present at high levels in resting normal M-B cells, while the transcript levels are lower in proliferating GC-B and in activated CD5+B lymphocytes. IL-2 production is limited to the actively proliferating GC-B blasts, IL-1 beta and IL-6 to resting M-B cells. The cytokine production profile of CD5+ malignant B-CLL cells differs from that of their putative normal counterparts and is more like the profile of M-B cells, since B-CLL cells produce IL-1 beta, TNF-alpha, TGF-beta, and IL-6. These observations lead to the following conclusions: among normal B lymphocyte populations, resting M-B lymphocytes are the most active cytokine producers, and B-CLL malignant B cells reflect the production pattern of normal resting B lymphocytes.