Jean Hurlimann

Immunohistochemistry in the differential diagnosis of liver carcinomas.

Immunohistochemical techniques were used to study 177 hepatic tumors (hepatocarcinoma, cholangiocarcinoma, hepatocholangiocarcinoma, adenocarcinoma of unknown origin, and metastatic carcinoma). Phenotypes suggestive of hepatocarcinoma included keratins 8 and 18, factor XIII a, α-fetoprotein, C-reactive protein, carcinoembry-onic antigen (CEA) cross-reacting antigen; those in effect that excluded hepatocarcinoma were keratins 1,5, 10, 11, 19, true CEA. C-reactive protein, used for the first time, proved to be a fairly sensitive and specific marker. Factor XIII a, which was thought to be synthesized only by histiocytes, was also present in hepatocytes. Immunohistochemistry appears to be an important tool in the diagnosis of hepatic tumors. As a result of this study, 32 cases were reclassified; several were found to be intermediate between hepatocarcinoma and cholangiocarcinoma. Sixteen cases apparently were true hepatocholangiocarcinomas. In 12 cases of hepatocarcinoma, some tumor cells expressed keratins of bile duct type. It was impossible to differentiate immunohistochemically cholangiocarcinoma from metastatic carcinoma, except in two cases with breast tissue markers.

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

SummaryT lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8− clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8− phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.

Cervical intraepithelial glandular neoplasia (adenocarcinoma in situ and glandular dysplasia): A correlative study of 23 cases with histologic grading, histochemical analysis of mucins, and immunohistochemical determination of the affinity for four lectins

Twenty‐three cases of cervical intraepithelial glandular neoplasia (CIGN)—a term encompassing adenocarcinoma in situ and glandular dysplasia of the uterine cervix—were studied histologically, histochemically for mucins (neutral mucins, sialomucins and sulfomucins), and immunohistochemically for the affinity of four lectins (WGA, PNA, RCA, UEA). For comparison, six cases of cervical invasive adenocarcinoma and ten cases of cervices without tumor were similarly studied. Criteria for histologic grading of CIGN into three degrees were proposed according to the hyperchromasia and the stratification of nuclei, number of mitoses, and amount of intracellular mucin. Two different types of CIGN were distinguished according to their histological aspect and their mucin pattern: CIGN type A, where the mucin pattern was qualitatively similar to that of normal endocervical mucosa, i.e., neutral mucins, sulfomucins and sialomucins; and CIGN type B, where the glandular cells resembled small intestinal goblet cells and the mucins consisted of neutral mucins and sialomucins with the absence of sulfomucins. Nine cases of CIGN were of type A, 2 of type B, and 12 of both type A and B. Differences in lectin binding existed between normal columnar cells, CIGN, and invasive adenocarcinomas, as well as between CIGN of type A and B. The intensity of the positive immunochemical reaction varied, as well as the type of binded lectin and its localization in the cell. There was a great heterogeneity in the same histologic group from one case to another, and even in the same case from one cell to another.

Desmin and neural marker expression in mesothelial cells and mesotheliomas.

The distribution of keratin, vimentin, desmin, muscular actin, S100, specific neuron enolase, and chromogranin was studied by immunoperoxidase staining in mesothelium, malignant mesotheliomas, and pulmonary carcinomas. The mesothelial cells were positive for keratin and vimentin on all smears of pleural and ascitic effusions; most of them were also positive for desmin but rarely for enolase and S100. None was positive for muscular actin. Sixteen malignant mesotheliomas expressed vimentin and keratin; six were also positive for desmin, three for desmin and neural markers, and five for neural markers. In comparison, none of the pulmonary carcinomas was positive for these markers. Mesothelial cells are able to express markers of divergent differentiation. Mesotheliomas also have such markers that are present in other malignant tumors and, in particular, in intra-abdominal desmoplastic small cell tumors with divergent differentiation. This entity and mesotheliomas probably originate from the same cell. Moreover, desmin, found in 56% of malignant mesotheliomas but absent in pulmonary carcinomas, may be useful in the differential diagnosis of these tumors.

Lymphomas and pseudolymphomas of the alimentary tract: An Immunohistochemical study with clinicopathologic correlations

One hundred one lymphoproliferative lesions of the gastrointestinal tract (66 malignant lymphomas, 20 pseudolymphomas, and 5 borderline lesions) were reviewed. The pathologic features were compared to the clinical findings, with reference to differential diagnosis and prognosis. Special attention was paid to immunohistochemical features. The gross appearance was diagnostic in only a limited number of cases. Endoscopic biopsy alone was also of limited value because only a diagnosis of probability could be made in several cases. In most of them the definitive diagnosis had to be based on histologic examination of the resected specimen. Immunohistochemical examination was found to be very useful as an ancillary diagnostic technique. Malignant lymphomas displayed either cytoplasmic immunoglobulins with a monoclonal pattern (47.1 per cent) or a negative reaction, whereas the pseudolymphomas were generally characterized by polyclonal immunoglobulins. Ninety per cent of malignant lymphomas with cytoplasmic immunoglobulins contained lambda chains. The survival probability was found to be related to the size of the lesion, the depth of infiltration, and the immunohistochemical characteristics of the tumors. The histologic type of the lymphomas was of limited value as a predictor of prognosis in the present series.

bcl-2 protein in invasive ductal breast carcinomas

The bcl-2 gene encodes a protein which inhibits programmed cell death (apoptosis). This protein was detected by immunohistochemical techniques in 48% of invasive ductal carcinomas of the breast. It was present in well-differentiated carcinomas with hormonal receptors, and proteins synthesized under the control of oestrogens: pS2, cathepsin D and ERD5. In contrast, bcl-2+ carcinomas are less frequently positive for p53 and have a Ki67 score under the mean. bcl-2 protects cells against apoptosis. Accumulation of p53 protein, which is indicative of p53 mutation, would have the same effect; however, these two proteins seem inversely related, an inverse correlation observed by others in breast cancer cell lines and in lymphomas. Tumours positive for bcl-2 escape apoptosis and have worse prognosis but this is not what is found; survival at 5 years, and particularly the absence of recurrence during the first 5 years after surgery, seem to be associated with bcl-2 positivity. The bcl-2 protein seems only to be an important prognostic factor in women over 54 years of age. Moreover, p53− bcl-2+ tumours have a better response to hormonal therapy than p53− bcl-2− tumours.

Immunoglobulins and secretory component in endometrium and cervix

The synthesis of immunoglobulins and secretory component in the cervix and the endometrium was studied by tissue culture and immunofluorescence. Out of the 75 cervical biopsies studied, 17 were epidermoid carcinomas, 8 were carcinomas in situ and 23 tissues had inflammatory or metaplastic lesions. A total of 49 samples from endometrium were studied, out of which 22 were in the proliferative phase, 17 were in the secretory phase and 4 were carcinomas. In the cervical tissues without lesions, there were very few plasmacytes, the synthesis of immunoglobulins was low and in 66% of the tissues the synthesis of IgG was equal to or higher than that of IgA. With local modifications, the IgG synthesis was even more preponderant and was very important in epidermoid carcinomas which were infiltrated with numerous IgG plasmacytes. Secretory component was synthesized by almost all the tissues except the epidermoid carcinomas. The endometrium did not synthesize immunoglobulins; secretory component was synthesized only by endometrial tissue in the secretory phase and by 2 of the 4 carcinomas studied. It seems that in the cervix and the endometrium there is no relationship between the production of secretory component and the presence of IgA plasmacytes which probably localise as a result of other influences. The conditions in which the local secretory immunological system would react preferentially remain to be determined.

Prognostic value of p53 protein expression in breast carcinomas.

A group of 196 ductal infiltrating carcinomas of the breast was examined immunohistochemically for p53. The purpose of this study was to show whether frozen and fixed tissues are equally adequate for detection of p53 and which antibodies should be used to have a prognostic value. Detection was superior on frozen to that on formalin-fixed tissues. It was not possible with any method to improve results on fixed tissues. Detection of p53 was different for each antibody: M 1801 detected 41 cases on frozen tissues, M-240 52 cases, M-421 28 cases. Using all the antisera, and the rabbit antiserum CM1, it was possible to detect 71 cases (36%). The percentage was the same in infiltrating lobular carcinomas but higher (94%) in medullary carcinomas. p53 was associated with high grade and ER-tumours. In formalin-fixed tissues, p53 had no prognostic value. In frozen tissues p53 was not an independent factor of prognosis. However, it was important in sorting out cases with bad prognosis in the ER-carcinomas and in the carcinomas without metastases. The prognostic value was different for each monoclonal antiserum. Positivity with M421 associated with negativity for M240, and positivity only for M1801 sorted out cases with a poor prognosis (67% and 50% deaths at 5 years).

Characterization of different amyloids with immunological techniques.

We studied 71 cases of amyloidosis from the autopsy material of our institute. 17 cases were secondary amyloidosis of which 7 had initially been diagnosed as primary amyloidosis; all cases reacted positively with an anti-substance A antibody; 13 showed a suppression and 4 a strong reduction of the Congo red staining following tissue incubation in KMnO4. 25 cases were identified as primary amyloidosis of which 7 had initially not been recognized as such. In 16 cases amyloid deposits reacted positively with antibodies specific for the light chains of the immunoglobulins (3 X kappa, 13 X lambda). A monoclonal plasmacytic cell proliferation in the bone marrow was seen in 14 cases. In all cases deposits were KMnO4 resistant, but 1 case showed a slight reduction of staining intensity. 27 cases were cardio-vascular (senile) amyloidosis; in 20 cases at least 3 organs showed deposits; 12 cases had deposits in 5 and more organs. 2 cases were heredofamilial amyloidosis. In those 29 cases deposits reacted positively with an anti-prealbumin antibody, but were negative for AA and the light chains of the immunoglobulins; the Congo red staining remained strong in all cases when previously incubated in KMnO4. KMnO4-Congo red staining and antisera specific for AA, L-chains and prealbumin proved of value for classification of amyloidosis and for its organ distribution.

Adenocarcinoma in situ and invasive adenocarcinoma of the uterine cervix. An immunohistologic study with antibodies specific for several epithelial markers

The distribution of carcinoembryonic antigen (CEA), secretory component (SC), fat globule membrane antigens (FGMA), and keratin was determined immunohistochemically in 22 invasive adenocarcinomas of various types and in 9 adenocarcinomas in situ of the uterine cervix. In the invasive adenocarcinomas 77% were positive for CEA, 47% for SC, 89% for keratin, and 77% for FGMA. In adenocarcinomas in situ 67% were positive for CEA, 11% for SC, and 44% for keratin. The location of the markers was variable in the cells, and the cells in a tumor were irregularly positive. For a given histologic type there were several phenotypes. No correlation was found between histologic types of invasive adenocarcinomas and the various phenotypes. It remains to be shown whether a particular phenotype has a particular biological behavior. The detection in the serum of the markers shown in histologic preparations could be useful in the postsurgical monitoring.