SUMMARYWe studied the distribution of the homeodomain proteins Pdx-1 and Nkx 6.1 in the developing rat pancreas. During early development, nuclear staining for both Pdx-1 and Nkx 6.1 occurred in most epithelial cells of the pancreatic anlage. Subsequently, Nkx 6.1 became more ␤ -cell-restricted, and Pdx-1 also occurred in other islet cell types and in the duodenal epithelium. During early pancreatic development, cells co-storing insulin and glucagon were regularly detected. The vast majority of these did not possess nuclear staining for either Pdx-1 or Nkx 6.1. Subsequently, cells storing insulin only appeared. Such cells displayed strongly Pdx-1-and Nkx 6.1-positive nuclei. Therefore, Nkx 6.1, like Pdx-1, may be an important factor in pancreatic development and in mature insulin cell function.

Exhaustive characterizations of antisera to the structurally related peptides pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY) enabled us to establish the developmental pattern of these peptides in rat and mouse pancreas. PYY was the earliest detectable peptide and was present in all early appearing endocrine cell types. NPY appeared later and occurred exclusively in a subpopulation of insulin cells, whereas PP cells arose latest. At the earliest stage studied, all endocrine cells stored PYY. Most of these cells also contained glucagon. Subsequently, the endocrine cells comprised glucagon+PYY cells and glucagon+PYY+insulin cells. Later, cells storing either only insulin or insulin+PYY appeared. Quantitations of the relative numbers of these cell populations during development were consistent with a precursor role of triple-positive (insulin+glucagon+PYY) cells. Moreover, bromodeoxyuridine (BrdU) injections at E15.5 showed that a large percentage of triple-positive cells were in S-phase and therefore were actively dividing, whereas almost no pure insulin cells or insulin+PYY cells synthesized DNA at this time. These results suggest that PYY-positive endocrine cells may represent precursors for mature islet cells.

HER2 is an erbB/HER type 1 tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. HER2 (c-erbB-2 or neu) is a member of the erbB/HER type 1 tyrosine kinase receptor family. This family of receptors mediates proliferation and differentiation in normal epithelial cells. Inappropriate over-expression may contribute to tumour growth and invasion. 1 Over-expression of HER2 has been demonstrated in many epithelial tumours, including breast cancer. 2,3 Over-expression of HER2 on the surface of a tumour cell makes this protein a potential target for the anti-HER2 monoclonal antibody (MAb) construct Herceptin. 4 Herceptin, when combined with cytotoxic chemotherapeutic agents, enhances their anti-tumour activity and significantly prolongs survival in metastatic breast carcinoma. 5,6 Potentially, non-small-cell lung cancer (NSCLC) tumours over-expressing HER2 may also be treated with Herceptin.Previous studies of HER2 expression in NSCLC varied in use of antibodies and immunohistochemical technique as well as the definition of positive cases. Studies on paraffin sections vary in data on membranous and cytoplasmic expression. Membranous staining has been demonstrated in 2% to 40% of NSCLC cases 7,8 and associated with a poor prognosis for adenocarcinoma. 8 Other studies have reported cytoplasmic immunostaining in 10% to 60% of NSCLC cases. 9 -13 Although only membranous expression would be expected to be functionally important, several of these studies have shown cytoplasmic HER2 expression, especially in adenocarcinoma, to be associated with a poor prognosis. 9 -11 One study has shown that NSCLC tumours containing higher levels of HER2 protein have a worse outcome. 14 Another study showed HER2 expression in 17/138 (12%) localised lung adenocarcinomas, with only 1 of these immunopositive cases demonstrating gene amplification. 15 This suggests that, unlike breast carcinoma, HER2 gene amplification in NSCLC is uncommon.The HercepTest was designed as a semi-quantitative immunohistochemical assay to determine HER2 protein over-expression in routinely processed breast cancer tissue, to aid the assessment of patients for whom HER2 antibody treatment with Herceptin is being considered. Cases with a HercepTest staining intensity of 2ϩ or 3ϩ may benefit from Herceptin therapy. 4,16,17 Our aim was to evaluate HER2 expression in NSCLC using the HercepTest and to correlate the results with clinicopathological findings and gene amplification. MATERIAL AND METHODSThis was a retrospect...

SUMMARYThe homeobox gene product Nkx 6.1 is of unknown function but is expressed in the pancreas and the antropyloric mucosa of the stomach. In the adult pancreas, Nkx 6.1 possesses an insulin cell-restricted distribution, whereas its localization in the stomach is unknown. We now show that the vast majority of serotonin-producing enterochromaffin cells of the antropyloric mucosa contain Nkx 6.1-immunoreactive nuclei. In addition, a subpopulation of cells co-storing serotonin and gastrin display Nkx 6.1-positive nuclei. Such cells have been postulated to represent precursors of mature gastrin and serotonin cells. The nuclei of the co-storing cells have previously also been found to be positive for another homeodomain protein, Pdx-1. Pdx-1-deficient animals were therefore investigated and were found to be devoid of Nkx 6.1-positive nuclei. Our data show that Pdx-1 is needed for Nkx 6.1 expression and suggest a role for Nkx 6.1 in the maturation of gastrin-and serotonin-positive precursor cells.

Insulin promoter factor-1 (IPF1) (renamed to pancreatic-duodenal homeobox factor-1, PDX1) was originally cloned and characterized as an islet beta-cell specific insulin gene transcription factor (1) and later shown to be essential for the formation of the mature pancreas (2, 3). In the adult normal pancreas PDX1 is almost exclusively expressed in the beta-cell compartment and generally absent from the alpha-cell while it is widely expressed in the pancreatic epithelium during development. Using pluripotent rat islet tumor cultures and derived insulinomas and glucagonomas we have analyzed differential expression of a large number of genes including the transcription factors PDX1, Nkx6.1, Pax6, and NeuroD. While NeuroD and Pax6 expression was detectable among all phenotypes, PDX1 was expressed in the pluripotent culture and maintained in the insulinoma, while Nkx6.1 was selectively co-induced with insulin during insulinoma formation. Both factors were not detectable in the glucagonoma. Nkx6.1 proved to have a highly beta-cell restricted expression in the adult rat. Forced expression of recombinant PDX1 in the glucagonoma resulted in efficient transcriptional activation of the endogenous insulin and IAPP genes, but did not affect glucagon gene activity. In this hybrid alpha/beta-cell phenotype the endogenous Nkx6.1 gene remained silent. We conclude that PDX1 in synergy with NeuroD specifies part of the beta-cell phenotype including transcriptional activation of insulin and IAPP genes, but that other factors such as Nkx6.1 and Pax6 are required for additional features of the fully mature beta-cell phenotype.

Koch, C., Jensen, S. S., 0ster, A. & Houen, G. A comparison of the immunogenicity of the native and denatured forms of a protein. APMIS 104: 115-125, 1996. The effect of heat denaturation on the physicochemical and immunological properties of a model protein, ovalbumin, and its formaldehyde/lysine-treated form was investigated. Polyacrylamide gel electrophoresis and gel filtration showed that heat denaturation converted ovalbumin to high Mr polymers, whereas formaldehyde/lysine-treated ovalbumin remained monomeric with only a small proportion forming oligomers. NMR analysis demonstrated that non-denatured structures could easily be differentiated from the denatured structures. Intraperitoneal immunization of rabbits and mice showed that both native and denatured forms of ovalbumin induced an immune response, but denatured forms of ovalbumin were found to be less immunogenic and to have a lower epitope density than native ovalbumin. Analysis of the antisera in crossed immunoelectrophoresis showed that they were specific for either native or denatured forms of ovalbumin. These findings were further investigated by ELISA and immunoaffinity chromatography, and the high specificity and low cross-reactivity was confirmed. We conclude that the immunogenic epitopes on denatured ovalbumin are different from those on ovalbumin, and that these epitopes reflect a continuum of denatured conformations.

Summary The findings in a patient who was found to have a sclerosing stromal tumour of the ovary associated with a tubal pregnancy are described. Measurement of hormones in blood before and after removal of the tumour suggested the tumour was andro‐gen‐secreting.

TO THE EDITOR: We read with great interest the joint recommendations from the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) concerning human epidermal growth factor receptor 2 (HER-2) testing in breast cancer. 1 The new recommendations are highly appreciated, and the validation, standardization, and accreditation initiatives are very important for increasing accuracy in HER-2 testing.The joint ASCO/CAP guideline is based on a comprehensive review of published data on HER-2 testing, except for the recommendation for changing the cutoff of stained cells from 10% to 30%. Most published data are based on results obtained using a US Food and Drug Administration (FDA) -approved test that defines a positive 3ϩ staining as being uniform, intensive membrane staining of more than 10% of invasive tumor cells. The new guideline recommends a different cutoff of 30% for 3ϩ. It is not clear whether there are data that support this new cutoff, and we recommend the publication of data from clinical trials to do so.As a point of correction, we wish to bring to your attention that Dako (Glostrup, Denmark) has a US FDA-approved assay, the HER2 FISH (fluorescence in situ hybridization) pharmDx Kit (Dako Denmark A/S). This kit is indicated as an aid in the assessment of patients for whom Herceptin treatment is being considered. 2 The cutoff stated in the package inserts of the approved tests will still be the legally valid cutoff, and it will be impossible to change it without presenting the US FDA with valid data that correlate to clin-ical outcome. We are concerned that the divergence between the recommendations in the package inserts of the approved HER-2 tests and the ASCO/CAP guideline concerning cutoff will generate confusion rather than clarification.