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We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolyposis colorectal cancer (HNPCC) patients using mutational and immunohistochemical analyses combined whenever possible with primer extension assays, designed to estimate hMLH1 and hMSH2 transcript expression in peripheral blood lymphocytes. Single-strand conformational polymorphism screening and PCR-direct sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, four amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or hMSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one additional case. The lack of hMLH1 or hMSH2 immunostaining was associated with the presence of microsatellite instability in the corresponding tumor and was also observed in tumors from patients negative for pathogenic mutations by mutational screening. There was a marked unbalance in the allelic expression of either hMLH1 or hMSH2 transcripts in three of eight unrelated HNPCC patients that could be analyzed, although a less marked unbalance was detected in two additional patients. Tumors from patients with germ-line unbalance in hMLH1 or hMSH2 transcript expression did not express the corresponding mismatch repair protein and displayed microsatellite instability. Our results indicate that constitutional alterations in hMLH1 and hMSH2 transcript expression may represent genetic markers for HNPCC carrier status also in cases in which mutational analysis did not detect a definite pathogenic variant. This suggests that transcript deregulation may represent a relevant mode of germ-line inactivation for mismatch repair genes.

Monoclonal antibodies (MAbs) COL-4 and COL-12, to the carcinoembryonic antigen (CEA), and B72.3, CC-49, CC-83, to the tumor-associated glycoprotein 72 (TAG-72), were used to study the expression of distinct epitopes of the two molecules in 71 cases of lung carcinoma of differing histotype. These MAbs reacted with the majority of adenocarcinomas by immunoperoxidase on tissue sections, but demonstrated a more restricted reactivity with squamous carcinomas. MAb CC-49 detected the highest percentages of adenocarcinoma cells while the B72.3 epitope was expressed more in squamous carcinoma cells. No significant reactivity with any of these MAbs was observed in small cell carcinomas. The expression of the CEA and TAG-72 epitopes in non-small cell lung cancers was highly heterogeneous: a distinct epitopes in non-small cell lung cancers was highly heterogeneous: a distinct epitope could be expressed by the majority of cells, whereas another of the same antigenic molecule was either poorly or not expressed. In adenocarcinomas, mixtures of anti-CEA, anti-TAG-72, and anti-(TAG-72 plus CEA) MAbs resulted in additive reactivity with an increase of the immunopositive tumors and of the percentages of immunostained cells. This was particularly evident for the anti-(TAG-72 plus CEA) mixture. In squamous cell carcinomas the increase was modest and was mainly related to anti-TAG-72 reactivity. These studies suggest variability in the antigenic structure of tumor-associated antigens expressed by carcinomas and indicate that anti-(TAG-72 plus CEA) mixtures may represent an immunological adjunct for clinical application in adenocarcinoma patients. On the other hand, TAG-72 should be considered a better target antigen, as compared to CEA, in the detection of squamous cell carcinomas.