Differences and homologies of chromosomal alterations within and between breast cancer cell lines : A clustering analysis

Background: The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. Results: A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. Conclusions: Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level. © 2014Rondón-Lagos et al.; licensee BioMed Central Ltd

Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues

P63 EXPRESSION AS A NEW PROGNOSTIC MARKER IN MERKEL CELL CARCINOMA.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin tumor, mostly affecting elderly individuals. Histogenesis of this rare tumor as well as prognostic criteria are a matter of dispute. Several prognostic indicators have been proposed for MCC. Gender (male), tumour size >5 cm or > 2 cm, location in the buttock, thigh, trunk or head & neck, advanced clinical stage, small cell size, high mitotic index, diffuse pattern as well as mutation of TP53 gene family, have all been reported as indicators of poor prognosis. The p63 protein is a member of the p53 family, and it can be frequently amplified and/or overexpressed in human tumours including carcinoma of head & neck, lung, skin, esophagus, mammary glands, urothelia, cervix, prostate glands, oral squamous cell carcinomas and in odontogenic tumours. Although p63 per se is not general indicator of poor prognosis, nevertheless it is often associated to poorly differentiated basaloid carcinoma. Recently Fukushima et al. suggested that p63 expression is a disadvantageous factor for prognosis in a subgroups of B-cell lymphomas. Our study will show that the same statement can be made in MCC. METHODS: An immunohistochemical analysis of markers of proliferation (Ki67/MIB1), neuroendocrine differentiation (chromogranin A and synaptophisin) and basal cell differentiation (p63) was performed in a series of 47 cases of MCC. Significance of pathological data and of immunoreactivity with different markers was evaluated by chi-squared test. Survival curves were calculated by Kaplan-Meyer method. The difference of survival was estimated using Wilcoxon or Mantel-Cox test. RESULTS: Immunohistochemical evidence of neuroendocrine differentiation and immunopositivity for cytokeratin 20 were observed in all cases, while p63 positivity (10 of neoplastic cell) was detected in approximately half (25/47; 53.2%) of the cases. Cases positive for p63 showed a more aggressive clinical course than the negative ones (Z =2.93; P=.0003; HR=22.22). CONCLUSIONS: Our data indicate that p63 expression is associated with a worse prognosis in MCC and it represents a new independent marker of clinical evolution with a better predictive power than the other prognostic parameters

MERITS OF THE PMiT (PAPILLARY MICROTUMOR) TERMINOLOGY IN THE DEFINITION OF INDICENDAL PAPILLARY CARCINOMAS OF THE THYROID: EXPERIENCE OF FIFTY CASES

Background: The term \u201cpapillary thyroid microcarcinoma (PTMC)\u201d is generally employed to designate an incidental papillary carcinoma measuring 1 cm or less witch manifests extremely indolent behaviour. PTCMs are often detected by chance in thyroids removed for benign clinical nodules or widespread processes. There has been an exponential increase in the detection of PTCMs thanks to the recent improvement and application of ultrasonography in the management of thyroid lesions. However, no definitive treatment guideline has been developed to indicate how best to treat and manage these small tumours. For this reason the surgeon and patients may become alarmed when the pathologist reports the presence of PTMC and this may lead to re-operation, radical dissection of the neck or extensive irradiation, all of which are deemed unnecessary. On the other hand, the pathologist is concerned in defining as a \u201ccarcinoma\u201d a lesions which he considers benign. For these reasons, Rosai et al proposed the term Papillary Microtumour of the thyroid (PMiT) during the 12th Annual Cancer Meeting in Porto (2003) and reported strict definition criteria for such entities. Methods: Since 2003, we have adopted, the Porto proposal criteria for PMiT in agreement with clinicians and surgeons. Here we report a series of 50 consecutive cases designed as PMiT (during the interval from March 2003 to August 2007) collected and treated at Molinette Hospital, University of Turin. Results: Patients (39F 11 M, median age 55.3 years) underwent to total thyrodectomy (47/50) or to lobectomy (3/50). No further treatment was performed. At histology, PMiT are often associated with benign nodules (19/50) or diffuse hyperplasic goitre (29/50), more rarely to parathyroid adenoma (1/50) and Thymoma B1 (1/50). All patients are alive and well after a median of 31.6 months follow-up (range from 5 to 57 months). Conclusions: In our experience this terminology, is well accepted by both clinicians and patients, since it decreases the danger of over-treatment, minimizes the psychological anxiety engendered by a diagnosis of carcinoma, and maintains the patient\u2019s eligibility for life insurance unalterated