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Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

Background and aims. The increasingly frequent use of mammography for the early diagnosis of breast cancer and the consequent identification of mammary lesions at a preclinical stage raises the fundamental problem of the differential diagnosis between non-suspected non-palpable lesions (NPL) which can therefore be monitored over time and suspected NPL or definite carcinoma requiring histological confirmation and surgical biopsy. The diagnostic accuracy of mammography alone is not sufficiently high to differentiate benign lesions from malignant or strongly suspected ones. The use of surgical biopsy in the event of suspected NPL could be significantly reduced by the use of stereotaxic cytology which would improve the diagnostic accuracy of mammography. Methods. The study refers to 72 suspected NPL undergoing surgical biopsy after having performed stereotaxic cytology on a sample taken with a dedicated mammographic device (Mammotest- TRC). Results. The rate of inadequate samples for correct cytological evaluation was 16.1%. Of the 72 NPL undergoing surgical biopsy, 40 (55.5%) were found to be carcinomas and 32 (44.5%) were benign lesions. The sensitivities of mammography alone and cytology alone in identifying infraclinical breast carcinoma were respectively 0.85 and 0.95. If the results of the two methods were evaluated together, the level of sensitivity was 0.98. Conclusions. The use of stereotaxic cytology enables a marked improvement to be achieved in the diagnostic accuracy of mammography for the identification of suspected NPL to undergo surgical biopsy, notably reducing the cost of biopsy (number of benign lesions for each carcinoma diagnosed) and consequent discomfort for patients.