c-erbB-2 protein expression was investigated immunohistochemically in frozen thyroid tissue specimens from 42 patients using a polyclonal sheep antibody. c-erbB-2 immunoreactivity was detected in 12 out of 17 papillary carcinomas, while no c-erbB-2 protein immunostaining was seen in cases of follicular adenoma (five cases), follicular carcinoma (five cases) or medullary carcinoma (one case), nor in cases of non-neoplastic tissue, including normal thyroid tissue from tumour-bearing glands. RNA was extracted from 51 thyroid tissue samples from 34 of the above patients, and c-erbB-2 mRNA was analysed by slot-blot hybridisation. c-erbB-2 mRNA was detectable in all samples, but papillary carcinomas and lymph node metastases showed significantly higher levels of c-erbB-2 mRNA compared to non-neoplastic tissue. The present demonstration of positive c-erbB-2 immunostaining in papillary thyroid carcinomas is contradictory to previous findings on formalin-fixed, paraffin-embedded material, and emphasises the importance of tissue quality for c-erbB-2 protein detection. Images Figure 1 Figure 3

Expanding knowledge, together with implementation of new techniques, has fuelled the area of translational medical research aiming at improving prognostication as well as prediction in cancer therapy. At the same time, new discoveries have revealed a biological complexity we were unaware of only a decade ago. Thus, we are faced with novel challenges with respect to how we may explore issues such as prognostication and predict drug resistance in vivo. While microarray analysis exploring expression of thousands of genes in concert represents a major methodological advancement, discoveries such as the finding of different mechanisms of epigenetic silencing, intronic mutations, that most gene transcripts in the human genome are subject to alternative splicing and that hypersplicing seems to be a tumour-related phenomenon, exemplifies a complex pathology that may not be explored with use of single analytical methods only. This paper discusses clinical settings for studying drug resistance in vivo together with a discussion of contemporary biology in this field. Notably, each individual parameter which has been found correlated to drug resistance in vivo so far represents either a direct drug target or a factor involved in DNA repair or apoptosis. On the basis of these findings, we suggest drug resistance may be explored on the basis of upfront biological hypotheses.

Summary The receptor-type oncogenes c-erbB2/neu and c-erbB have been found amplified and/or overexpressed in a number of tumours of epithelial origin. We have studied the expression of oncogenes in biopsies from human thyroid tumours. The c-erbB2/neu and c-erbB oncogenes showed two-to three-fold higher levels of RNA in papillary carcinomas and lymph node metastases as well as in one adenoma when compared to non-tumour tissue. The nuclear oncogenes c-myc and c-fos were found to be expressed at varying levels in both non-tumour and tumour tissue. RNA transcripts specific for the platelet-derived growth factor A and B chains and the N-ras oncogene were detected in one anaplastic carcinoma. Neither rearrangements nor amplifications of oncogenes were observed in the thyroid tumours. These data are particularly interesting in light of the recent findings that epidermal growth factor induces proliferation and dedifferentiation of normal thyroid epithelial cells in vitro. We suggest that the epidermal growth factor or other ligands for the c-erbB and c-erbB2/neu receptors may contribute to the development and/or maintenance of the malignant phentotype of papillary carcinomas of the thyroid.

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Background: Mutations in TP53 and its upstream p53-activator Chk2, are known to be associated with resistance to anthracycline and mitomycin treatment in locally advanced breast cancer. However, this association is not fully predictive, as some tumours are therapy resistant despite harbouring wild-type TP53 and CHK2. ATM is an upstream activator of both p53 and Chk2. Here, we explored ATM mutational status and expression levels with respect to anthracycline/mitomycine resistance in locally advanced breast cancers having primary chemotherapy with doxorubicin or 5FU-mitomycin and previously analyzed for TP53 and CHK2 status. Methods: In order to investigate ATM's potential predictive role, we performed MLPA-analysis, qPCR on ATM mRNA, ATM promoter methylation analyses and complete sequencing of the entire ATM coding region in patient biopsies from two prospective studies on resistance to doxorubicin and 5-FU/mitomycin in locally advance breast cancer (n=36 and n=38, respectively). Results: We performed complete sequencing of the ATM coding exons in 74 patients including 17 patients revealing primary resistance to chemotherapy. ATM mutations were detected in tumours from 5 patients. No mutation was detected among tumors resistant to chemotherapy. In addition, we observed 9 polymorphic variants present in more than one tumour, affecting or not affecting the amino acid sequence of the ATM protein (n = 34 patients in total). No correlation was observed between polymorphism status and response to therapy. No methylation of the ATM promoter, large deletions or duplication/amplifications of the ATM gene was observed. ATM expression levels varied substantially, with a ratio of 49 between the highest versus lowest level recorded. Among patients with no mutations in TP53 or CHK2, low ATM expression levels were significantly correlated to lack of response to doxorubicin or 5-FU/mitomycin (p=0.023; Mann-Whitney test). In contrast, ATM-levels were not suppressed in non-responding tumors harbouring TP53 or CHK2 mutations (p=0.642; Mann-Whitney test). Defining ATM mRNA levels in the lower 50%, 33% or 25% of the patient cohort to be reduced, tumors with either reduced levels of ATM or mutations affecting TP53 or CHK2 revealed a high risk for therapy resistance (p=0.004, p=0.001 and p=0.006, using the different ATM-level cut-offs; Fischer exact test). Interestingly, in a similar prospective study with a smaller number of patients displaying progressive disease upon treatment with epirubicin (n = 10), we find 2 out 5 TP53 and CHK2-wild-type non-responding tumors to express very low levels of ATM (within the lowest 5% of the cohort). Conclusions: Our data suggests that low expression of ATM may substitute for TP53 and CHK2 mutations causing resistance to anthracycline-and mitomycin therapy in breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-02.