Licia Laurino

Calretinin: A novel immunocytochemical marker for mesothelioma

Immunohistochemistry is a powerful diagnostic adjunct in the differential diagnosis between malignant mesothelioma (especially of the epithelial type) and adenocarcinoma metastatic to the serous membranes. Most of the immunological probes commonly used, however, recognize antigens expressed by the epithelial malignancies and absent from mesothelial cells and mesotheliomas. Probes suitable for the positive identification of mesotheliomas are comparatively scarce and much less commonly used because of their reduced sensitivity and specificity, their unsuitability for staining routinely fixed and embedded tissues, or their lack of commercial availability. We now document that two different polyclonal antisera to calretinin consistently immunostain mesothelial cells and malignant mesotheliomas both in routinely fixed and embedded tissue sections and in cytological preparations of serous effusions. The diagnostic sensitivity of this novel immunocytochemical approach reached 100%, allowing immunostaining of all 44 mesotheliomas investigated, which included five biphasic and three sarcomatoid types. The specificity of calretinin immunoreactivity was checked against 294 adenocarcinomas of different origin (19 serosal metastases and 275 primary tumors potentially able to metastatize to serosal membranes) relevant for the discussion of the differential diagnosis with malignant mesothelioma: only 28 cases showed focal immunoreactivity for calretinin. We conclude that calretinin is a most useful marker for the positive identification of malignant mesotheliomas.

Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody

FLI-1 nuclear transcription factor has been proposed as a useful tool in the differential diagnosis of small round cell sarcomas. Recently, FLI-1 has been reported as the first nuclear marker of endothelial differentiation. However, its clinical use has been hampered by major interpretation problems, due to the presence of background staining as well as staining variation between different lots of the same antiserum. In this study, a novel monoclonal antibody raised against the carboxyl terminal of the FLI-1 protein (clone GI146-222, BD Pharmingen) was tested in a series of small round cell and vascular neoplasms. Furthermore, in order to assess FLI-1 specificity, we analyzed its expression in a series of common epithelial and nonepithelial malignancies. In total, 15 Ewings sarcomas, 10 rhabdomyosarcomas, 5 desmoplastic small round cell tumors, 10 synovial sarcomas, 10 high-grade pleomorphic sarcomas, 10 malignant melanomas, 5 Merkels carcinomas, 10 colonic adenocarcinomas, 10 breast carcinomas, 10 lung adenocarcinomas, 20 angiosarcomas, 5 epithelioid hemangioendotheliomas, 10 Kaposis sarcomas and 10 benign hemangiomas, were stained. A strong FLI-1 immunoreactivity was detected in all Ewings sarcomas and vascular neoplasms, highlighting the high sensitivity of FLI-1 monoclonal antibody. However, 2/5 Merkels carcinomas and 1/10 malignant melanomas showed a strong nuclear immunostaining, suggesting that FLI-1 may not be so helpful in the differential diagnosis of cutaneous Ewings sarcoma. In addition, a weak immunoreactivity was found in 3/5 Merkel cell carcinomas, 3/10 synovial sarcomas, 5/10 malignant melanomas, 6/10 lung adenocarcinomas and in 1/10 breast carcinomas. In contrast, all the rhabdomyosarcomas, desmoplastic small round cell tumors, high-grade pleomorphic sarcomas and colonic adenocarcinomas tested were negative. Importantly, in contrast with previous studies, no background staining was observed. Our results indicate that FLI-1 monoclonal antibody can be reliably applied to the differential diagnosis of small round cell neoplasms of soft tissue, and confirm its important role as nuclear marker of endothelial differentiation, mainly helpful in those cases in which technical artifacts are seen by using the traditional membranous and cytoplasmic endothelial markers.

The prevalence of BCL-2 immunoreactivity in breast carcinomas and its clinicopathological correlates, with particular reference to oestrogen receptor status.

BCL-2 protein plays a pivotal role in overriding programmed cell death (apoptosis), thus favouring a prolonged survival of normal and neoplastic cells. Expression of the bcl-2 gene has been documented in some human tumours (non-Hodgkins lymphomas and prostatic adenocarcinomas), but findings in breast carcinomas have not been reported. We have used the monoclonal antibody 124 to investigate BCL-2 expression in 212 breast carcinomas, and to correlate it with the oestrogen (ER), progesterone (PR) and epidermal growth factor receptor (EGFR) status, and with other clinicopathological variables including tumour type, grade, stage, growth fraction (as evaluated by Ki-67 immunostaining), and p53 accumulation. Of the 212 carcinomas, 173 (81.6%) exhibited BCL-2 immunoreactivity in more than 25% of the neoplastic cells. BCL-2 immunoreactivity was strongly correlated with ER and PR expression (P<0.00001), with the lobular type (P=0.012) and with better differentiated neoplasms (P=0.00003), whereas it was inversely correlated with EGFR (P<0.00001), p53 (P=0.0004) and Ki-67 (P=0.0002) immunoreactivities. No association was found with tumour stage (T and N categories). We conclude that bcl-2 expression in breast cancers is related to the oestrogen-dependent transcription pathway.

p21/WAF1/CIP1 EXPRESSION IN NORMAL MUCOSA AND IN ADENOMAS AND ADENOCARCINOMAS OF THE COLON: ITS RELATIONSHIP WITH DIFFERENTIATION

The immunoreactivity of p21 was evaluated in normal mucosa and in adenomas and adenocarcinomas of the human large bowel. In normal mucosa, p21 immunoreactivity was seen in the superficial third of the crypts (maturation compartment) and in surface (terminally differentiated) epithelium, and was mutually exclusive with Ki67/MIB1 reactivity. These data show that p21 expression is inversely related to proliferation and directly related to terminal differentiation. In adenomas, p21‐reactive cells were frequently clustered in the superficial areas and were non‐reactive for MIB1. In adenocarcinomas, p21 staining was heterogeneous: high p21 expression (19 cases) was associated with lower stage and lack of p53 overexpression. p21‐reactive cells were devoid of MIB1 immunoreactivity, but no relationship could be found between p21 and MIB1 labelling indices. p21 heterogeneity may be related to alterations in the p53‐dependent induction pathway: high p21 expression was associated with low to absent p53 reactivity, with presumed normal p53 function; low p21 expression was associated with p53 overexpression, with presumed p53 alteration resulting in loss of function.

Rabbit Monoclonal Antibodies A Comparative Study Between a Novel Category of Immunoreagents and the Corresponding Mouse Monoclonal Antibodies

Rabbit monoclonal antibodies (RabMAbs) represent a novel category of immunoreagents that may combine the best properties of both mouse monoclonal antibodies (MMAs) and of rabbit antisera. In the attempt to verify the performance of this new class of antibodies on paraffin-embedded tissue, RabMAbs against estrogen receptor, progesterone receptor, Ki-67, cyclin D1, CD3, CD5, CD23, and synaptophysin were tested on several tumor types as well as normal tissues. The results were compared with those obtained with classic MMAs against the same antigens. RabMAbs appear to offer increased sensitivity with no apparent loss of specificity. On routine use they permit higher working dilutions (5 to 10 times on average), allowing significant improvement in terms of laboratory efficiency. The robustness of RabMAbs is further proved by the fact that in some instances optimal staining can be obtained even without antigen retrieval. In consideration of the high performance observed, routine use of RabMAbs may contribute significantly to standardize diagnostic immunohistochemical procedures.

p53 protein expression in non-neoplastic lesions and benign and malignant neoplasms of soft tissue.

Alterations of the p53 tumour suppressor gene, with consequent nuclear p53 protein accumulation, are among the most common genetic lesions in human neoplasms. In the present paper we show p53 immunoreactivity in 65% of malignant and 21% of intermediate malignancy soft tissue tumours, and in 48% of benign/reactive soft tissue lesions. p53 immunoreactivity of sarcomas can be interpreted as an indirect indication of a mutation of the corresponding p53 gene, suggesting that its alteration may have a role in their pathogenesis. Our data on p53 immunoreactivity in benign lesions of the soft tissues are among the first demonstrations of p53 over‐expression in benign/reactive conditions. We cannot exclude mutations of the p53 gene in these cases, but it is difficult to sustain this hypothesis in reactive/pseudoneoplastic lesions. Alternatively p53 immunoreactivity in benign processes could be due to an increase in wild‐type p53 as a result of different physiological mechanisms (cell type‐specific p53 regulation, cell maturation, DNA repair). Our results do not indicate that immunohistochemical demonstration of p53 expression is a marker of malignancy in soft tissue tumours and therefore is of limited use in differential diagnosis. However, they suggest the need for further molecular genetic studies in order to elucidate the biological significance of the abnormal expression of p53 in benign soft tissue lesions.

Natural history of imatinib-naive GISTs: a retrospective analysis of 929 cases with long-term follow-up and development of a survival nomogram based on mitotic index and size as continuous variables.

Gastrointestinal stromal tumor (GIST) natural history per se has not been extensively investigated yet, with most data being drawn from large studies with a relevant referral bias. Hence, the estimation of prognosis still remains a critical issue. We retrospectively evaluated 929 GISTs resected between 1980 and 2000 in 35 Italian institutions. A total of 526 patients were found to be suitable for refining risk assessment through the development of a survival nomogram. Median follow-up was 126 months. On testing for potential prognostic parameters, age, tumor site, size, and mitotic index proved to be predictors of OS on both univariable and multivariable Cox model analyses, whereas necrosis and cytonuclear atypia were significant on univariable analysis only. The discriminative ability of the model, including the parameters selected after a backward procedure (C=0.72), improved compared with the National Institutes of Health 2002 (C=0.64) and the National Comprehensive Cancer Network 2007 (C=0.63). On the basis of these data we developed a prognostic nomogram for survival that considers site, size, and mitotic index as continuous variables, providing estimates stratified for patients aged ⩽65 and >65 years. This nomogram is a tool based on survival. It overcomes problems that result from artificial categorization of continuous variables. We believe that in the future this should also be attempted by nomograms based on the risk of relapse.

Rabbit Monoclonal Antibodies

Rabbit monoclonal antibodies (RabMAbs) represent a novel category of immunoreagents that may combine the best properties of both mouse monoclonal antibodies (MMAs) and rabbit antisera. In the attempt to verify the performance of this new class of antibodies on paraffin-embedded tissue, RabMAbs against estrogen receptor, progesterone receptor, Ki-67, cyclin D1, CD3, CD5, CD23, and synaptophysin were tested on several tumor types as well as normal tissues. The results were compared with those obtained with classic MMAs against the same antigens. RabMAbs appear to offer increased sensitivity with no apparent loss of specificity. On routine use they permit higher working dilutions (5 to 10 times on average), allowing significant improvement in terms of laboratory efficiency. The robustness of RabMAbs is further proved by the fact that in some instances optimal staining can be obtained even without antigen retrieval. In consideration of the high performance observed, routine use of RabMAbs may contribute significantly to standardize diagnostic immunohistochemical procedures.

Relationship between cirrhosis, liver cancer, and hepatic metastases. An autopsy study

Consecutive autopsies (5241) performed in the Trieste area and consecutive autopsies (6511) performed in the Tokyo—Chiba area were analyzed to study the frequency of liver metastases in cirrhotics. The Italian material included 500 cases and the Japanese material included 529 cases of liver cirrhosis. Both of these groups were matched for sex and age with a control group. The results were similar in both areas and confirmed the widely held but disputed opinion that metastases in cirrhotic liver are rare. These results seem to be clinically important since they agree with the fact that most neoplasms in cirrhotic liver are primary. From a biologic and epidemiologic point of view, these results call for reconsideration of the complex relationship existing between cancer and liver cirrhosis, in relation to major causative factors such as alcohol and hepatitis B virus (HBV) infection.

KP1 (CD68) expression in benign neural tumours. Further evidence of its low specificity as a histiocytic/myeloid marker

KP1 (CD68) is a monoclonal antibody raised against a lysosomal fraction of human lung macrophages. and is suggested to be a useful marker for recognition of true histiocytic neoplasms and of the distribution of macrophages in both reactive and neoplastic processes’. KP1 can be expressed in both normal and neoplastic non-haemopoietic tissue^^-^. Its detection within the lysosome-rich cells of granular cell neoplasms5 may indicate a lysosome-directed specificity rather than the expression of true histiocytic lineage. As neural differentiation of granular cell tumours is widely accepted, a series of benign neural lesions has been investigated in order to further study the range of distribution of KP1.