Annalisa Chiriatti

Cytoskeleton and paclitaxel sensitivity in breast cancer : the role of β-tubulins

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the β subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I β‐tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel‐binding site of β‐tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of β‐tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC50. The Class I β‐tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF‐7 and SK‐BR‐3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III β‐tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III β‐tubulin could at least partially increase paclitaxel‐chemosensitivity. The hypothesis of a relationship between β‐tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel‐based chemotherapy. Thirty‐five percent (95% CI: 45–31) of patients with high Class III β‐tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III β‐tubulin tumor expression could be considered a predictive biomarker of paclitaxel‐clinical resistance for breast cancer patients.

Topoisomerase‐I, thymidylate synthase primary tumour expression and clinical efficacy of 5‐FU/CPT‐11 chemotherapy in advanced colorectal cancer patients

While several studies have reported that thymidylate synthase (TS) tumour expression can be a reliable predictive marker of clinical response to 5‐Fluorouracil (5‐FU) for advanced colorectal cancer patients, only a few studies that searched for predictive factors of irinotecan (CPT‐11) clinical response are available. The aim of the present study has been to verify the predictive value of immunohistochemical topoisomerase‐I (Topo‐I) and TS primary tumour expression in a consecutive series of 62 advanced colorectal cancer patients that received a first line 5‐FU/CPT‐11 chemotherapy. TS and Topo‐I immunostaining was observed in 76% and 43% of tumours, respectively, resulting in a significant relationship within each tumour (r=0.365, p<0.004). Patients with different TS tumour expression showed a similar percentage of Objective Clinical Response, OR (40% vs. 28% of OR in low and high TS‐expressing tumours, respectively, p=ns); also, patients with different Topo‐I tumour expression did not show a different probability of OR (39% vs. 29% of OR in high and low Topo‐I expressing tumours, respectively; p=ns).The tumour expression of these 2 biomarkers also did not impact on time to progression and overall survival of patients. Furthermore, the combined analysis of TS and Topo‐I tumour status did not permit to individualize subgroups of patients with different probability of OR. With multivariate analysis, only patient Performance Status significantly impacted on OS (Hazard ratio 4.87; p=0.02) of these patients. We can conclude that high TS tumour expression seems not to preclude a clinical activity for 5‐FU/CPT‐11 polichemotherapy in advanced colorectal cancer patients; furthermore, clinical response and prognosis of colorectal cancer patients treated with 5‐FU/CPT‐11 regimen do not differ in tumours with different TS or Topo‐I expression.

Biological role of NHERF1 protein expression in breast cancer.

Aims:  To determine the role of Na+/H+ exchanger regulatory factor (NHERF1) in breast cancerogenesis and progression.

Genetic heterogeneity by comparative genomic hybridization in BRCAx breast cancers

The chromosomal changes in eight familial BRCAx breast cancers (i.e., negative for BRCA1 or BRCA2) were analyzed by comparative genomic hybridization (CGH) to investigate intratumor heterogeneity. This was the first step in a study of most frequent chromosomal aberrations in BRCAx familial breast cancers. Laser microdissection analysis of paraffin tissue samples was followed by whole-genome amplification. CGH was performed on DNA isolated from two to three different cell groups per case to detect any cytogenetic aberrations in important clones that might have been missed when analyzing DNA extracted from large numbers of cells. The results were compared, to evaluate the influence of tumor heterogeneity on CGH, and the heterogeneity was confirmed comparing CGH with fluorescence in situ hybridization results. Different chromosomal aberrations were detected between adjacent clones within the same section, which highlights the utility of microdissection in addressing the problem of heterogeneity in whole-genome studies. Some chromosomal regions were more frequently altered in the eight BRCAx tumors; loss of 2q, 3p, 3q, 8p, 9p, and 15q and gains of 1p, 4p, 4q, 5p, 6q, 12q, and 19p were the most common. Further studies focusing on specific genes and sequences with more sensitive approaches, such as array-CGH, are warranted to confirm these findings.

Touch imprint cytology in tumor tissue banks for the confirmation of neoplastic cellularity and for DNA extraction.

CONTEXT Learning the characteristics of frozen tissue samples stored in tumor banks for biological studies remains a problem. OBJECTIVE To assess the use of touch imprint cytology on fresh tissue samples as a rapid and reliable method of determining the presence and quantity of neoplastic cells before freezing. DESIGN Touch imprint cytology was performed on 259 specimens of operable breast cancer. Touch imprints were prepared from fresh tissue specimens before freezing samples for storage. Each tumor sample was imprinted on a glass slide and stained with hematoxylin-eosin. Tumor cellularity was quantified as negative, poor, moderate, or rich. RESULTS A significant correlation was found between samples with a tumor size greater than 2 cm and high tumor cellularity (P = .03; chi(2) test). Furthermore, 35% of ductal tumors showed higher tumor cellularity compared with lobular tumors (P < .001; chi(2) test). No association was found between lymph node status and tumor grade. When samples for which more than 2 imprints were available were examined, tumor cellularity among imprints of the same sample showed an overall agreement of 0.67 (P < .001; kappa statistic). It was also determined that the higher the cellularity, the higher the agreement. Our data also showed concordance of 0.87 (P < .001; kappa statistic) between touch imprint cytology imprints and histologic sections from contiguous tumor. Moreover, 11 randomly selected samples underwent DNA extraction, polymerase chain reaction, and sequencing to verify the feasibility of DNA analyses. We found that DNA from touch imprint cytology was amplifiable and suitable for direct sequencing. CONCLUSIONS Touch imprint cytology may represent an important step in the quality control of tumor cellularity of breast cancer specimens designed to be stored in tumor biobanks and a valid method for assessing the suitability of such tissue for further biomorphologic and biomolecular applications.