Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

A synonymous EGFR polymorphism predicting responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients.

Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild-type patients benefit from that treatment. In this study, we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11 % of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG + AA genotypes. Taken together, our findings could be used to better define CRC populations responding to anti-EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness.

The story of poorly differentiated thyroid carcinoma: From Langhans' description to the Turin proposal via Juan Rosai.

Rosai, reinterpreting Langhans' "proliferating goiter," adopted the term "poorly differentiated carcinoma" for a specific thyroid tumor with insular features 30 years ago. This tumor type is only one of those approached by Dr. Rosai in the thyroid field (a PubMed search as of August 31, 2015 on "Rosai & thyroid" disclosed 73 articles), but seems the most innovative and representative of his heavy contribution to thyroid tumor classification. The diagnostic problems associated with PDTC recognition date back a long time, with a still ongoing debate on the nature of PDTC, its morphological diagnostic features, its clinical significance, and its optimal therapeutic approach. In 2004, PDTC was at last incorporated in the WHO classification of thyroid tumors, but the proposed diagnostic criteria were heterogeneous, controversial, and hardly applicable in the diagnostic practice. A consensus conference held in Turin in 2006 was lead by the authors and Dr. Rosai and confirmed the presence of geographical differences among claimed classical PDTC forms, which were responsible for a poor interobserver reproducibility of the diagnostic criteria. A diagnostic algorithm was therefore designed to define the crucial parameters to categorize PDTC and better stratify these distinctly aggressive tumors.

Critical roles of specimen type and temperature before and during fixation in the detection of phosphoproteins in breast cancer tissues.

The most efficient approach for therapy selection to inhibit the deregulated kinases in cancer tissues is to measure their phosphorylation status prior to the treatment. The aim of our study was to evaluate the influence of pre-analytical parameters (cold ischemia time, temperature before and during tissue fixation, and sample type) on the levels of proteins and phosphoproteins in breast cancer tissues, focusing on the PI3 kinase/AKT pathway. The BALB-neuT mouse breast cancer model expressing HER2 and pAKT proteins and human biopsy and resection specimens were analyzed. By using quantitative reverse phase protein arrays (RPPA), 9 proteins and 16 phosphoproteins relevant to breast cancer biology were assessed. Cold temperatures before and during fixation resulted in a marked improvement in the preservation of the reactivity of biological markers (eg, ER, HER2) in general and, specifically, pHER2 and pAKT. Some phosphoproteins, eg, pHER2 and pAKT, were more sensitive to prolonged cold ischemia times than others (eg, pS6RP and pSTAT5). By comparing the phosphoprotein levels in core needle biopsies with those in resection specimens, we found a marked decrease in many phosphoproteins in the latter. Cold conditions can improve the preservation of proteins and phosphoproteins in breast cancer tissues. Biopsies ≤ 1 mm in size are the preferred sample type for assessing the activity of deregulated kinases for personalized cancer treatments because the phosphoprotein levels are better preserved compared with resection specimens. Each potential new (phospho)protein biomarker should be tested for its sensitivity to pre-analytical processing prior to the development of a diagnostic assay.

The pre-analytical phase in surgical pathology.

Several sequential passages are involved in the pre-analytical handling of surgical specimens from resection in the surgical theater to paraffin-embedding and storage. Each passage is highly critical and can significantly affect the preservation of morphology, antigens, and nucleic acids. Some key points in this process are still undefined and are subject to high variability among hospitals. High quality and standardization are demanded and pathologists should therefore work to comply with all novel clinical requests (such as genomic and antigenic testing for targeted molecular therapies). Under-vacuum sealing of surgical pieces can be a safe and reliable alternative to storage in large formalin-filled boxes; it prevents dehydration and favors cooling by removing air. Moreover, it implements tissue banking and preservation of nucleic acids. After transport of specimens to pathological anatomy laboratories, the next passage, fixation, has been the object of several attempt to find alternatives to formalin. However, none of the substitutes proved successful, and formalin fixation is still considered the gold standard for preservation of morphology and antigens. RNA has instead been found to be heavily affected by degradation and fragmentation in formalin-fixed tissues. Based on the hypothesis that RNA degradation would be inhibited by maintaining a low temperature, a protocol based on processing tissues with formalin at low temperature (cold fixation) was evaluated and proved useful in obtaining a reduction in RNA fragmentation. Finally, the problem of storage is discussed, in order to find ways to guarantee feasibility of molecular analyses even years after the original diagnosis.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue.

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

Presence of osteoinductive factors in bovine colostrum.

New approaches in the treatment of skeletal defects may benefit from the use of soluble biological factors. We previously standardized a derivative of bovine colostrum (SBCD), deprived of casein and fat and rich in cytokines. In the present study, we tested its possible use as an adjuvant in bone healing. SBCD contained factors involved in stromal cell stimulation and differentiation and induced cytokine production from stimulated mesenchymal stem cells (MSCs). In vitro, SBCD promoted proliferation, migration and, in association with osteogenic factors, osteogenic differentiation of osteoblastic and MSCs. In in vivo experiments of subcutaneous Matrigel injection in mice, SBCD plus hydroxyapatite, but not hydroxyapatite nor SBCD alone, induced recruitment of macrophages and stromal cells. After 60 days, plugs containing SBCD and hydroxyapatite were densely calcified and diffusely positive for osteocalcin, supporting the occurrence of an early osteogenic process. These results indicate that SBCD is a rich source of factors with osteoinductive properties.

"To be or not to be in a good shape": diagnostic and clinical value of nuclear shape irregularities in thyroid and breast cancer.

Variation in both nuclear shape and size ("pleomorphism"), coupled with changes in chromatin amount and distribution, remains the basic criteria for microscopy in a cytologic diagnosis of cancer. The biological determinants of nuclear shape irregularities are not clarified, so, rather than on the genesis of nuclear irregularities, we here focus our attention on a descriptive analysis of nuclear pleomorphism. We keep in mind that evaluation of nuclear shape as currently practiced in routine preparations is improper because it is indirectly based on the distribution of DNA as revealed by the affinity for basic dyes. Therefore, over the last years we have been using as criteria morphological features of nuclei of thyroid and breast carcinomas as determined by immunofluorescence, in situ hybridization, and 3D reconstruction. We have translated this approach to routine diagnostic pathology on tissue sections by employing immunoperoxidase staining for emerin. Direct detection of nuclear envelope irregularities by tagging nuclear membrane proteins such as lamin B and emerin has resulted in a more objective definition of the shape of the nucleus. In this review we discuss in detail methodological issues as well as diagnostic and prognostic implications provided by decoration/staining of the nuclear envelope in both thyroid and breast cancer, thus demonstrating how much it matters "to be in the right shape" when dealing with pathological diagnosis of cancer.

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study.

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Telocytes are the common cell of origin of both PEComas and GISTs: an evidence-supported hypothesis.

We advance the hypothesis that the telocyte might be the cell of origin of both PEComas (perivascular epithelioid cell tumours) and GISTs (gastro-intestinal and extra-gastrointestinal stromal tumours). The hypothesis is supported by data from the literature reporting that both PEComas and GISTs, as well as telocytes, share the expression of several markers. These data were supplemented by original immunohistochemical tests on selected series. Specifically: (1) Melanoma markers (Melan A, MiTF) typical of PEComas are expressed by a substantial fraction of GISTs. A fraction of GISTs was also found positive for CD63, a tetraspanin protein originally described in melanomas and marking exosomes. (2) c-KIT (CD117), proper of the vast majority of GISTs, can be expressed by PEComas (as well as by telocytes). (3) Markers described in telocytes (CD34, S-100, smooth muscle actin and vascular endothelial growth factor) have been reported as positive in cases of PEComas and GISTs. Telocytes show distinctive ultrastructural features with thin, extended, telopodes and are likely involved in inter-cellular signalling via paracrine secretion as well as by shed vesicles and exosomes. These cells have been described in many locations (cavitary and non-cavitary organs) and might display potentialities of a wide spectrum of differentiation (and function). In conclusion we propose that telocytes could be the common cells of origin for both PEComas and GISTs.

Expression of p63 is the sole independent marker of aggressiveness in localised (stage I-II) Merkel cell carcinomas.

Merkel cell carcinoma of the skin is a malignant neuroendocrine tumour, whose prognostic criteria are a matter of dispute. Specifically, no predictor is presently available in stage I-II tumours. We collected clinical and follow-up data from 70 Merkel cell carcinomas of the skin. The same cases were studied for p63 expression by immunohistochemistry, by reverse-transcription PCR (RT-PCR) and TP63 gene status by FISH and for presence of Merkel cell polyomavirus by PCR. Stage emerged as a significant prognostic parameter (P=0.008). p63 expression, detected in 61% (43/70) of cases by immunohistochemistry, was associated with both decreased overall survival (P<0.0001) and disease-free survival (P<0.0001). Variable expression patterns of the different p63 isoforms were found only in cases immunoreactive for p63. In these latter lesions, at least one of the N-terminal p63 isoforms was detected and TAp63α was the most frequently expressed isoform. TP63 gene amplification was observed by FISH in only one case. Presence of Merkel cell polyomavirus DNA sequences was detected in 86% (60/70) of Merkel cell carcinomas and did not emerge as a significant prognostic parameter. Merkel cell carcinoma cases at low stage (stage I-II) represented over half (40/70 cases, 57%) of cases, and the clinical course was uneventful in 25 of 40 cases while 15 cases died of tumour (10/40 cases) within 34 months or were alive with disease (5/40 cases) within 20 months. Interestingly, a very strict correlation was found between evolution and p63 expression (P<0.0001). The present data indicate that p63 expression is associated with a worse prognosis in patients with Merkel cell carcinoma, and in localised tumours it represents the single independent predictor of clinical evolution.

Androgen receptor expression is a significant prognostic factor in estrogen receptor positive breast cancers.

The purpose of this article is to evaluate the prognostic value of androgen receptor (AR) expression in patients with estrogen receptor (ER)-positive breast cancer, treated with endocrine therapy, with or without the addition of chemotherapy. A consecutive series of 953 patients with ER-positive breast cancer, treated between 1998 and 2003, was selected. Repeated immunohistochemistry confirmed the expression of ER in the tumor of 938 patients. AR expression was measured by immunohistochemistry. The Kaplan-Meier method, logrank test and multivariate Cox models were used to explore the impact of AR expression on time to relapse (TTR) and disease specific survival (DSS) in all patients and in subgroups treated with chemo-endocrine therapy or endocrine therapy alone. AR immunoreactivity was assessable in 859 tumors and positive in 609 (70.9%). AR expression was a significant marker of good prognosis for TTR (P = 0.001) and DSS (P < 0.001). This effect was particularly evident in the group of patients receiving chemo-endocrine therapy (TTR (P = 0.015) and DSS (P < 0.001)). Cox models confirmed AR as an independent variable for both TTR (P = 0.003, HR 0.444, 95%CI 0.258-0.765) and DSS (P < 0.001, HR 0.135, 95%CI 0.054-0.337). Thus, we focused on ER-positive luminal B breast cancer that may be selected for chemotherapy because of their more aggressive immunophenotype. In this subset AR expression identified a group of patients with better prognosis for TTR (P = 0.017, HR 0.521, 95%CI 0.306-0.888) and DSS (P = 0.001, HR 0.276, 95% CI 0.130-0.588). AR expression is an independent prognostic factor of better outcome in patients with ER-positive breast cancers.

Poorly differentiated carcinoma of the thyroid: validation of the Turin proposal and analysis of IMP3 expression.

The Turin Proposal algorithm defines poorly differentiated carcinoma on the basis of the presence of solid/trabecular/insular growth pattern, absence of conventional nuclear features of papillary carcinoma, and the presence of at least one of the following features: convoluted nuclei, mitotic activity > or =3/10 HPF, or tumor necrosis. IMP3 appears to have diagnostic and prognostic value in many solid tumors, including thyroid carcinomas. We examined a series of follicular-cell carcinomas with prominent solid patterns diagnosed at Mayo Clinic (56 cases) (Rochester, MN, USA) and at the University of Turin (96 cases) (Northern Italy) to validate the Turin consensus criteria defining poorly differentiated carcinoma of the thyroid and to evaluate the prevalence and prognostic behavior of this tumor. On this series, we analyzed the expression of conventional markers by immunohistochemistry and we investigated the expression of IMP3 by both immunohistochemistry and qRT-PCR. The prevalence of poorly differentiated carcinoma among the USA cases was 1.8% (56/3128) and that in the cases of Northern Italy was 6.7% (96/1442). Tumor characteristics were similar in the cases from the USA and from Italy except for extensive vascular invasion and a prevalent insular growth pattern (lower the former, higher the latter in the Italian series). In univariate analysis, the risk of death was higher for age > or =45, tumors > or =4 cm, and IMP3 positivity. Multivariate analysis showed that the risk of death from poorly differentiated carcinoma was higher for age > or =45. The Turin consensus criteria can reliably select poorly differentiated carcinomas. Tumors from the USA and from Italy showed similar overall survival, although the prevalence of poorly differentiated carcinoma was higher in Northern Italy. Expression of IMP3 appears to be an adverse prognostic factor for poorly differentiated thyroid carcinoma.

Mixed micropapillary-ductal carcinomas of the breast: a genomic and immunohistochemical analysis of morphologically distinct components.

Micropapillary carcinomas (MPCs) can present as a rare histological special type of breast cancer; however, this histological type is more frequently found admixed with invasive ductal carcinomas of no special type (IDC-NSTs). We have previously demonstrated that pure MPCs constitute a distinct entity at the morphological and genetic levels. Here, we sought to determine whether mixed MPCs have genomic aberrations similar to those found in pure MPCs, and to investigate whether the distinct morphological components of MPCs harbour different genetic aberrations. Using high-resolution microarray comparative genomic hybridization (aCGH), we profiled a series of 10 MPCs of mixed histology and 20 IDC-NSTs matched for grade and oestrogen receptor (ER) status. In addition, we generated tissue microarrays containing a series of 24 pure and 40 mixed MPCs and performed immunohistochemical analysis with ER, progesterone receptor (PR), Ki-67, HER2, cytokeratin (CK) 5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1 and E-cadherin antibodies. In situ hybridization was employed to evaluate the prevalence of HER2, TOP2A, EGFR, CCND1, MYC and FGFR1 gene amplification. Our results demonstrate that mixed MPCs harbour similar patterns of genomic aberrations and phenotype (82.5% luminal and 17.5% HER2) compared to pure MPCs. A comparison between the distinct morphological components of mixed MPCs in a pairwise fashion revealed that both components harbour strikingly similar genomic profiles. When compared to grade- and ER-matched IDC-NSTs, mixed MPCs significantly more frequently harboured amplification of multiple regions on 8q (adjusted Fisher's p value < 0.05). Furthermore, mixed MPCs displayed higher proliferative rates than grade- and ER-matched IDC-NSTs. Our results suggest that micropapillary differentiation in breast cancer may identify a subgroup of more aggressive ER-positive breast carcinomas, even in those featuring a mixed histology, and that mixed MPCs are more closely related to pure MPCs than to IDC-NSTs.

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis.

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Neuroendocrine differentiation in breast cancer: established facts and unresolved problems.

Neuroendocrine breast carcinoma (NEBC) diagnosis relies on (i) presence of morphologic neuroendocrine features, and (ii) neuroendocrine markers expressed in more than 50% of tumor cells. The World Health Organization classification describes 3 main histologic types: the solid, the small/oat cell, and the large cell variant. In addition, we have recently proposed a further categorization into 5 subgroups: the first 3 categories encompass solid lesions and include (i) solid cohesive carcinomas, (ii) alveolar carcinomas, and (iii) small cell carcinoma; the last subgroups include mucin-producing tumors which are (iv) solid papillary carcinomas and (v) cellular mucinous carcinomas. Chromogranin A and synaptophysin have been considered as the most sensitive and specific neuroendocrine markers in NEBC. At the molecular level, recent gene expression profiling studies have shown that NEBCs pertain to the luminal molecular type, being positive for hormone receptors and negative for HER2. Moreover, it has been demonstrated that mucinous and neuroendocrine carcinomas are transcriptionally distinct from conventional invasive ductal carcinomas. Following the above criteria, NEBCs constitute approximately 1% of all breast carcinomas. The clinical effect of neuroendocrine breast cancer is still a matter of debate; however, when compared with unselected breast cancers, NEBCs show a less aggressive clinical behavior.

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue.

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT-PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step-sectioning protocol at 100 micron-intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT-PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT-PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT-PCR negative cases, metastases were detected only on sections distant from those analysed by RT-PCR. CEA and/or CK19 were positive by RT-PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.

Anti-angiogenic properties of calcium trifluoroacetate.

Endothelial cell proliferation and the formation of new vessels are strictly regulated by angiogenic factors (e.g., VEGF) that induce the activation of signal transduction pathways controlled by calcium dynamics. Using in vitro and in vivo experiments, we investigated the effect of calcium trifluoroacetate (CaTFAc), a complex, poorly dissociated salt that is characterized by its low toxicity, on angiogenesis. In vitro, CaTFAc inhibited VEGF-induced effects on endothelial cell proliferation. In two in vivo models of angiogenesis, a Matrigel plug in mice and a chick embryo chorioallantoic membrane, CaTFAc inhibited the VEGF-induced formation of new vessels. The exact mechanism of action is still under investigation, but in vitro experiments demonstrate that CaTFAc induced a reversible increase in the levels of intracellular calcium under basal conditions and prevented calcium signaling induced by VEGF. These results are the first to suggest that CaTFAc may be useful for the treatment of diseases caused by enhanced angiogenesis.