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Aims: HER2 protein is over-expressed in 15-30% of breast carcinomas. Immunohistochemistry (IHC) is a common and inexpensive method able to specifically detect HER2 protein. However, lack of standardization of IHC has been considered responsible for discrepancies in HER2 status assessment performed by IHC and fluorescence in-situ hybridization (FISH). This prompted us to perform a multicentric IHC calibration test to achieve a maximum accuracy of HER2-IHC compared with HER2-FISH taken as the reference method., Methods and Results: Twelve French laboratories participated in this study, including 119 cases of invasive breast carcinomas for which both fixed and frozen tissues were available. HER2 expression was determined in fixed tissues by individual in-house IHC techniques, using either CB11 (Novocastra, Newcastle, UK) or A0485 (Dako, Glostrup, Denmark) anti-HER2 antibodies. Two cut-off values were used: 10% and 60% of immunostained cells. In 116 of the 119 cases, HER2 gene status could also be determined by FISH on frozen sections, performed in a single laboratory. Results were centralized and compared. When suboptimal concordance between IHC and FISH was observed, IHC was calibrated and a second run was performed. The specificity, sensitivity and accuracy of IHC compared with FISH were noted before and after calibration. Forty-four out of 116 (38%) tumours showed HER2 gene amplification. Accuracy of IHC was complete in the first run for 6/12 laboratories. Calibration, necessary for the six others, relied mainly on the combination of a heat-induced epitope retrieval step with an increase of dilution of the primary antibody. In the second run, HER2 over-expression was found in 46 (40%) and 44 (38%) of the 116 cases, using 10% or 60% of stained cells as cut-offs, respectively. The corresponding accuracy rates were 93% and 95%., Conclusions: This study showed that a high accuracy of IHC could be obtained for the determination of HER2 status in all laboratories using their in-house IHC technique, provided that a calibration process was performed. Antigen retrieval procedure, high dilutions of anti-HER2 antibody and the use of specific controls were crucial for HER2-IHC calibration. A 95% accuracy rate of IHC, using FISH as gold standard, was obtained by considering immunolabelling HER2-IHC results as a continuous variable, and taking 60% invasive stained cells as the cut-off for HER2 over-expression.

Human papilloma virus (HPV) DNA sequences (HPV types 16, 18, 33, 35 or uncharacterized) were detected by Southern blot hybridisation and polymerase chain reaction in 84% of 106 early-stage invasive carcinomas of the uterine cervix. Among HPV-positive patients, the risk of overall relapse did not differ with individual HPV types. Compared with HPV-positive patients, those with no detectable HPV DNA had a 2.6 times higher risk of overall relapse (p less than 0.05) and 4.5 times higher risk of distant metastases (p less than 0.01). The 24-month relapse-free survival rate in HPV-positive patients was significantly higher than that in HPV-negative patients (77% vs 40%), and the difference was similar (91% vs 56%) among those who were node-negative. These data indicate that HPV-negative cervical carcinomas may represent a biologically distinct subset of tumours that carry a poorer prognosis than do HPV-positive cancers.

Plasma fibronectin was determined in cancer patients and in age- and sex-matched controls and analyzed as a function of age, size of tumor, receptor content of the tumor, metastases and treatment. In the control population, plasma fibronectin increased with age exponentially. The age-dependent increase in plasma fibronectin was strongly attenuated in the cancer population. As normal and cancer curves intersect at about 40-46 years, below this age cancer plasmas have slightly higher values than normal, above this age the inverse is true. No correlation was found between estrogen or progesterone receptor levels and plasma fibronectin values, nor with plasma albumin. Tumor patients with distant metastases gave slightly but significantly higher values than those with local or no metastases. No significant difference was found between tumors when Bloom grading was taken as the second parameter instead of age. The size of the tumor or the type of treatment had no influence. Increased proteolytic activity, increased trapping of plasma fibronectin in tissues and especially in the stromal (desmoplastic) reaction and/or modifications in plasma fibronectin biosynthesis may well be responsible for these results.

The ultrastructure of 47 primary breast cancers was studied. For each tumour, the characteristics of approximately 200 malignant cells were examined. Eleven features were scored from 1 to 3 and multivariate analysis showed that seven of these could be used to define an ultrastructural index of differentiation. Differentiation at the ultrastructural level was associated with the presence of steroid receptors. Differentiated tumoral cells contained oestrogen receptor and progesterone receptor in 81.8% and 66.7% of cases, respectively. Poorly differentiated cells contained oestrogen receptors in 50% and progesterone receptor in 14.3% of cases. Comparison of histological grading by the method described by Scarff, Bloom & Richardson with the ultrastructural index of differentiation showed a rather loose correlation which was not significant in this group of 47 patients. The authors conclude that differentiation at tissue or cellular levels yields differing information, only the latter being closely correlated with steroid receptor presence.