Prognostic Role of p53 Immunohistochemical Status in invasive Breast Cancer. A Retrospective Review of 1387 Cases With Luminal-Like/Her2 Negative Breast Tumors.

PURPOSE: The aim of this study was to evaluate the prognostic role of p53 immunohistochemical (IHC) expression in a large cohort of patients with hormone receptors (HR)-positive/Her2-negative primary invasive breast cancer. METHODS: Retrospective review of consecutive cases treated at our Breast Unit between 2003 and 2013. Patients were divided into 3 subgroups based on p53 IHC expression: null (0%), low (0.1%-49%), and high (≥50%) p53 expression. RESULTS: A total of 1387 patients were included in the study with a median follow-up of 86 months. After adjusting for age, size, node status, lymphovascular invasion, progesterone, and Ki67 expression, only null p53 immunophenotype was associated with worse disease-free survival (DFS) (OR 1.74, 95% IC, 1.11-2.71, P = .015) and distant recurrence-free survival (DRFS) (OR 1.73, 95% IC, 1.04-2.90, P = .036). Null p53 impacted significantly DFS and DRFS also in patients with early breast cancer. CONCLUSIONS: p53 IHC expression affects survival and, thus can be a valuable tool in the management of patients with HR-positive/Her2-negative breast cancer.

Polarization states and far-field optical properties in dielectric photonic crystal slabs.

We study the role of topological singularities like Bound States in a Continuum (BICs) or Circularly Polarized States (CPSs) in determining ellipticity of the far-field polarization in dielectric metasurfaces. Using finite-difference time-domain as well as rigorous coupled-wave analysis simulations, we determine the behavior of the Stokes parameter S3 in the whole k space above the light cone, with special regard to the region close to the singularities. Moreover, we clarify the relation between the topological singularities and the circular dichroism in reflectivity.

Doubly-Resonant Photonic Crystal Cavities for Efficient Second-Harmonic Generation in III-V Semiconductors.

Second-order nonlinear effects, such as second-harmonic generation, can be strongly enhanced in nanofabricated photonic materials when both fundamental and harmonic frequencies are spatially and temporally confined. Practically designing low-volume and doubly-resonant nanoresonators in conventional semiconductor compounds is challenging owing to their intrinsic refractive index dispersion. In this work we review a recently developed strategy to design doubly-resonant nanocavities with low mode volume and large quality factor via localized defects in a photonic crystal structure. We built on this approach by applying an evolutionary optimization algorithm in connection with Maxwell equations solvers; the proposed design recipe can be applied to any material platform. We explicitly calculated the second-harmonic generation efficiency for doubly-resonant photonic crystal cavity designs in typical III-V semiconductor materials, such as GaN and AlGaAs, while targeting a fundamental harmonic at telecom wavelengths and fully accounting for the tensor nature of the respective nonlinear susceptibilities. These results may stimulate the realization of small footprint photonic nanostructures in leading semiconductor material platforms to achieve unprecedented nonlinear efficiencies.

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft.

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches.

Mitochondrial DNA analysis efficiently contributes to the identification of metastatic contralateral breast cancers.

PURPOSE: In daily practice, a contralateral breast cancer (CBC) is usually considered as a new independent tumor despite the indications of several studies showing that the second neoplasia may be a metastatic spread of the primary tumor. Recognition of clonal masses in the context of multiple synchronous or metachronous tumors is crucial for correct prognosis, therapeutic choice, and patient management. Mitochondrial DNA (mtDNA) sequencing shows high informative potential in the diagnosis of synchronous neoplasms, based on the fact that somatic mtDNA mutations are non-recurrent events, whereas tumors sharing them have a common origin. We here applied this technique to reveal clonality of the CBC with respect to the first tumor. METHODS: We analyzed 30 sample pairs of primary breast cancers and synchronous or metachronous CBCs with detailed clinical information available and compared standard clinico-pathological criteria with mtDNA sequencing to reveal the metastatic nature of CBCs. RESULTS: MtDNA analysis was informative in 23% of the cases, for which it confirmed a clonal origin of the second tumor. In addition, it allowed to solve two ambiguous cases where histopathological criteria had failed to be conclusive and to suggest a clonal origin for two additional cases that had been classified as independent by pathologists. CONCLUSION: Overall, the mtDNA-based classification showed a more accurate predictive power than standard histopathology in identifying cases of metastatic rather than bilateral breast cancers in our cohort, suggesting that mtDNA sequencing may be a more precise and easy-to-use method to be introduced in daily routine to support and improve histopathological diagnoses.

Preoperative computed tomography assessment for a deep inferior epigastric perforator (DIEP) flap: a new easy technique from the Bologna experience.

BACKGROUND: Deep inferior epigastric perforator (DIEP) flap reconstruction is the gold standard reconstructive technique for women undergoing breast cancer surgery. A preoperative computed tomography angiography (CTA)-dedicated protocol and 3D reconstructions are mandatory for correct surgical planning. PURPOSE: To evaluate the diagnostic performance of a new preoperative CTA protocol and a new reconstruction method in the assessment of DIEP technique. MATERIAL AND METHODS: A total of 263 women (median age 49 years, age range 26-73 years) underwent preoperative CTA examination before DIEP flap breast reconstruction. A CTA-dedicated protocol followed by 3D-reconstructions were performed. Identification, branching pattern, and caliber at origin were assessed for each perforator. Intraoperative findings were the standard of reference. The sensitivity, positive predictive value, and diagnostic accuracy of the preoperative CTA protocol were calculated. RESULTS: In 255/263 (97%) patients, the dominant perforators assessed by CTA resulted adequate for surgical reconstruction. In 260/263 (99%) cases, the imaging localization of the dominant perforators corresponded with those seen intraoperatively (mean errors ≤1 cm). The preoperative CTA imaging sensitivity, positive predictive value, and diagnostic accuracy in determining the localization of perforators were 99% (95% CI 98-100), 100% and 99% (95% CI 98-100), respectively. No statistically significant differences were found between the CTA findings and the surgical findings for the assessment of branching pattern and caliber of the dominant perforators (P < 0.001). CONCLUSION: The present protocol has demonstrated high accuracy in the CTA imaging assessment of the perforators before DIEP flap reconstruction with high reproducibility between CT and surgical findings.

Hereditary breast cancer: screening and risk reducing surgery.

BACKGROUND: The screening modalities for women at high risk for breast cancer has received an increasing role during the last years. The aim of this study was to evaluate the performance of our screening program comparing the diagnostic sensitivity of clinical breast examination, mammography, ultrasonography (US) and magnetic resonance imaging (MRI). METHODS: Clinical Breast examination, mammography, US and MRI for each patient with BRCA1 and BRCA2 mutation who underwent breast surgery in our Institution from October 2008 to April 2016 were retrospectively evaluated. The diagnostic accuracy for MRI and for the other surveillance tests in identifying early breast cancer were assessed. RESULTS: Twenty-six female patients with genetic mutation underwent breast surgery. Twenty-two out of 26 (85%) developed cancer during the dedicated screening protocol whereas 4 women who underwent surgery did not have cancer. Imaging was able to detect cancer in all 22 patients (per patient sensibility of 100%), identifying all 35 neoplastic lesions (per lesion sensibility of 100%). The combination of Clinical Breast Examination, US and mammography aided the cancer diagnosis in 14 (64%) of patients with a sensitivity of 64% and specificity of 100%. MRI identified all the cancers, with sensibility and specificity of 100%. Moreover, in 8 (36%) of the 22 patients who developed breast cancers, the cancers were detected only by MRI, revealing a significant superiority respect to the other surveillance modalities (P<0.05). CONCLUSIONS: MRI demonstrated to be the best imaging modality in detection of breast cancer even for lesion <1 cm. Prophylactic mastectomy is the most effective risk reduction strategy in women at high risk, contributing to the reduction of anxiety related to the condition of a carrier.

Recurrent periductal mastitis: Surgical treatment.

BACKGROUND: Recurrent periductal mastitis is a benign breast disorder that often features a mammary fistula that runs between periareolar skin and the ductal mammary system. Due to the high recurrence rate of this disease, its management is controversial. This study was designed to assess the efficacy of fistulectomy (Hadfield operation), particularly with regard to its long-term outcome. METHODS: We reviewed all women with recurrent periductal mastitis who underwent the Hadfield operation in the Breast Center in S.Orsola-Malpighi Hospital (Bologna University) from 2005 to 2015. All but one of the patients were heavy smokers and presented with a recurrent periareolar abscess and a periareolar mammary fistula. RESULTS: Eighteen women underwent the Hadfield surgical treatment. Mean age at the time of presentation was 42 years; 17 of 18 women smoked >10 cigarettes/d. All patients had a breast ultrasonography or mammography. Half of the patients had undergone antibiotic therapy with one or more prior abscess drainages or another form of operative treatment. All patients who underwent operative treatment had no postoperative events and were satisfied with the cosmetic results. Squamous metaplasia was always present in the specimens. After a median follow-up of 36 months, 2 patients developed a recurrence after a few months; neither had stopped smoking. CONCLUSION: Based on our review of the literature and taking into account the results of this study, it seems clear that the best treatment involves a combined total excision of the affected duct and the fistulous tract. Due to the important role of smoking in this disease, it is important to encourage patients to stop smoking.

Has breast cancer in the elderly remained the same over recent decades? A comparison of two groups of patients 70years or older treated for breast cancer twenty years apart.

OBJECTIVES: Breast cancer (BC) in the elderly population is by far the most frequent malignancy in Western countries; however, little evidence is available regarding the specific management of this group. The purpose of this study was to identify how the biological and clinical characteristics of cancer have changed over the past 20years by comparing two groups of elderly patients with breast cancer operated on 20years apart. The secondary endpoint was to underline potential changes in surgical strategy over the past 20years. MATERIALS AND METHODS: One group of consecutive elderly patients undergoing surgery for BC between January 1990 and December 1993 (Group A), and one group undergoing surgery between January 2008 and December 2011 (Group B) were identified and analyzed. Data regarding surgical treatment, stage, tumor grading, hormonal and HER2/neu receptors, and Ki-67 were collected and compared. RESULTS: A total of 422 elderly patients underwent surgical treatment, 142 in Group A and 280 in Group B. An earlier stage at presentation was detected in Group B, T1 (57.5% B vs. 31.6% A) and N0 (64.6% B vs. 54.2% A). Surgical treatment in the first group was more extensive while conservative procedures were more frequently performed in the second group. Despite the earlier presentation, tumor grade was higher in Group B (G3 10.6% A vs. 32.1% B, p<0.05). Overexpression of Ki-67 was again more frequent in Group B (56.2% B vs. 32.5% A, p<0.05). Hormonal and HER2/neu receptor expression was comparable. CONCLUSIONS: Nowadays, elderly patients with BC are more likely to present at an early stage; therefore, conservative surgery is a feasible option. Despite potential bias related to changes of pathology and immunohistochemistry examination techniques over the decades, the biological characteristics of recent patients with BC seem to be consistent with more aggressive tumors. Tailored treatment should be offered with regard to biological age, the cancer-specific profile and active life expectancy.

Effectiveness of sentinel lymph node intraoperative examination in 753 women with breast cancer: are we overtreating patients?

OBJECTIVE: The goal of our study was to evaluate the sensitivity and specificity of sentinel lymph node biopsy (SLNB) frozen section (FS) examinations to detect metastatic lymph nodes and also its potential role in avoiding unnecessary demolitive axillary surgery. BACKGROUND: SLNB is the standard of care in surgical oncology of the breast. Intraoperative evaluation of the SLN seems to achieve sufficient sensitivity for macrometastasis (Ma), leading to axillary lymph node dissection (ALND) only when strictly necessary. Is it equally as clear when to perform ALND if micrometastasis (Mi) or isolated tumor cells (ITCs) are detected? METHODS: All consecutive patients from January 2005 to September 2010 operated on for breast cancer were prospectively enrolled. All patients underwent an FS SLNB. The sensitivity and specificity of SLN FSs in detecting Ma, Mi, and ITCs was calculated. All patients with Ma or Mi at FS underwent ALND. For all patients who underwent ALND, the number of metastatic non-SLNs was recorded and correlated to the size of the SLN metastasis. RESULTS: A total of 753 patients were enrolled. FS examination had an overall 54% sensitivity and 100% specificity in detecting metastatic disease (Ma/Mi/ITCs). The sensitivity rises to 89% if only Mas were considered and to 64% if Mas and Mis were counted together. All patients with Mas or Mis detected at FS had a completion ALND during the same procedure (156/222). All patients with Mas detected at final pathology (16 false negatives, 2.6%) and 50 women with Mis or ITCs (119 false negatives, 20%) underwent a delayed ALND. When Mis or ITCs were detected in the SLN, only 8 of 73 (10.9%) and none of 4 (0%) patients, respectively, had at least 1 metastatic non-SLN after ALND. Two patients (2/460, 0.43%) who had negative SLNs showed local axillary recurrence. After a mean follow-up of 32 months, none of the 71 patients with Mis or ITCs who did not undergo a second operation showed local recurrence. CONCLUSIONS: SLNB FS is highly effective in detecting the subgroup of patients who may benefit from completion ALND during the same surgical procedure. The role of Mi/ITCs in the SLN(s) is still unclear, but our data lean toward a less aggressive surgical approach.

The Neoadjuvant Net: a patient- and surgeon-friendly device to facilitate safe breast-conserving surgery in patients who underwent neoadjuvant treatment.

The primary goal of the study was to describe an innovative and helpful tool in defining the minimal surgical margins necessary during breast-conserving surgery (BCS) after neoadjuvant treatment: the Neoadjuvant Net (NN). The secondary endpoint was to assess its usefulness in achieving postoperative disease-free margins and reducing Ipsilateral Breast Tumor Recurrences (IBRTs). The breast-conserving surgical technique together with the use of the Neoadjuvant Net is herein reported. Age, stage at diagnosis, clinical and pathological response, lymph node status, type of surgery, margin status, and incidence of local and distant recurrence were retrospectively analyzed. Seventy-five patients underwent BCS following medical treatment from 2000 to 2011. The majority of the patients had significant size reduction (63/75, 84%). Twenty-two had a complete clinical response but only 11 (11/75, 14.7%) showed a complete pathological response. Two patients (2/75, 2.67%) had infiltrated surgical margins. After a mean follow-up of seventy months, 3 patients (3/75, 4%) had IBRTs and 4 women had distant metastases (4/75, 5.34%). The NN is an easy-to-use, non-invasive instrument designed with the purpose of facilitating the surgeon's task of reducing infiltrated margins and IBTRs.

IL23R, NOD2/CARD15, ATG16L1 and PHOX2B polymorphisms in a group of patients with Crohn's disease and correlation with sub-phenotypes.

Recent genomic research has identified interleukin-23 receptor (IL23R), nucleotide-binding oligomerization domain containing 2 caspase-activation recruitment domain 15 (NOD2/CARD15), autophagy related 16-like 1 (ATG16L1) and paired-like homeobox 2b (PHOX2B) as susceptibility loci for Crohn's Disease (CD). Our aim was to investigate these gene variants in a group of CD patients and to analyse the correlation to sub-phenotypes such as gender, smoking habits, disease behaviour at diagnosis, severity of disease and extra-intestinal manifestations. Nineteen patients with CD and 20 healthy controls were included in the study. The gene variants IL23R rs7517847 and rs11209026, NOD2/CARD15 rs2066845, PHOX2B rs16853571, ATG16L1 rs2241879 and rs2241880 were genotyped by PCR followed by sequencing. The frequency of the G risk allele of IL23R rs7517847 was found to be increased in patients with CD (42%) compared to that in control subjects (20%) [odds ratio (OR), 2.9; 95% confidence interval [CI], 1.06-7.9; P=0.03]. In addition, the homozygous condition GG was also associated with CD (OR, 8.70; 95% CI, 0.9-81.6; P=0.038). The analysis of correlation of genotype to sub-phenotypes showed an association of ATG16L1 rs2241879 with the lack of extra-intestinal manifestations (OR, 0.03; 95% CI, 0.002-0.45; P=0.006), and the patients defined as non-smokers displayed an increased frequency of the risk allele C (P=0.03). The present study confirms the association of the heterozygous and homozygous IL23R rs7517847 variant with CD and suggests an additive effect of smoking to the ATG16L1 rs2241879 C risk allele SNP, in the context of the multifactorial model established for the development of CD and a protective effect of the same allele against extra-intestinal manifestations.

Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.

Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the controls. Our data confirm the extreme sensitivity of RT-PCR, making this technique able to detect minimal amounts of mRNA expressed in a non-tissue-specific manner. Moreover, DGED remains a powerful tool to identify candidate epithelial markers in blood, such as colon related mRNAs. However, to date, none of these qualified as tumour markers.

Nipple discharge: is its significance as a risk factor for breast cancer fully understood? Observational study including 915 consecutive patients who underwent selective duct excision.

Nipple discharge (ND) is a common symptom seen in breast cancer clinics. The primary aim of this study was to identify preoperative risk factors for breast cancer in patients with pathologic ND. The secondary aim was to assess the clinical and pathological effectiveness of physical examination, galactography, cytological examination of the discharge, selective duct excision and ductoscopy. All patients operated on between 1975 and 2008 who presented with ND as their only symptom was analyzed. Discharge's characteristics, cytological data and galactography reports were recorded. The relationship between each individual finding and the risk of breast cancer was calculated. For each diagnostic tool, the sensitivity, specificity and complication rates were calculated and compared. Nine-hundred-fifteen patients underwent selective duct excision. Two-hundred-nineteen patients (23.9%) were found to be affected by carcinoma. In 100/330 (30.3%) patients with bloody discharge and in 42/239 (17.6%) patients with serous secretion cancer was detected (P = 0.004, P = 0.013, respectively). Patients with sero-sanguinous or coloured discharge had the same risk of cancer as the population analyzed (23.9%, P = NS). Galactographic finding of irregular stenosis seemed to be associated with a higher risk of cancer (P = 0.0001). Cytological findings C5 and C4 were associated with cancer (P = 0.001). Selective duct excision showed highest sensitivity and specificity. In conclusion, the well established role of bloody secretion is confirmed. The supposed benign aetiology of serous, coloured or sero-sanguinous discharge is questionable. The high specificity of the cytological exam justifies routine examination of the ND. Selective duct excision can be considered as the diagnostic gold-standard.

An easy-to-use solution for clinical audit in colorectal cancer surgery.

BACKGROUND: Clinical audit has been increasingly required for the accreditation process in every modern healthcare system. Data collection and analysis are excessively time-consuming in everyday practice. The primary aim of our study was to evaluate the effectiveness of an innovative database to assist surgeons in monitoring clinical practice outcomes in colorectal cancer surgery. The second purpose was to compare observed mortality rates to 3 risk-predicting operative scoring systems. METHODS: Data were evaluated from 208 consecutive patients undergoing elective and emergency surgery for colorectal cancer over a 2-year period (2003-2004). A new database was developed with specific queries to compare the observed and the expected mortality rates according to 3 scoring systems: the Portsmouth-Physiological and Operative Severity Score for enUmeration of Mortality and morbidity (P-POSSUM), the ColoRectal-Physiological and Operative Severity Score for enUmeration of Mortality and morbidity (CR-POSSUM), and the Association of ColoProctology or Great Britain & Ireland (ACPGBI) score. Results were discussed at regular intervals. Surgeons' satisfaction with each system was evaluated with a questionnaire. RESULTS: The observed mortality rate was 6.25%, which was significantly lower than the values predicted by CR-POSSUM and ACPGBI colorectal scores (9.14% and 19.42%, respectively; P < .05). P-POSSUM was the most accurate predictor of mortality, with a value of 7.93%. A total of 80% of the surgical staff considered this type of surgical audit activity as clinically useful. CONCLUSION: The study confirms the usefulness of a dedicated database in a surgical audit activity. The ACPGBI colorectal score largely overestimated 30-day mortality in our experience.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. METHODS: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. RESULTS: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. CONCLUSION: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer.

BACKGROUND: The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. METHODS: In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. CONCLUSION: The design of new approaches to identify such markers is warranted.

Search for epithelial-specific mRNAs in peripheral blood of patients with colon cancer by RT-PCR.

Research has widely supported the efficacy of screening for colorectal cancer in reducing mortality. A blood-based assay potentially represents a more accessible early detection tool for the identification of solid tumor cells originating from a primary tumor site in the body. We demonstrate a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as markers of malignancy in blood samples of patients with colon cancer. The present study aims to identify a set of specific mRNAs expressed in epithelial cells but not in blood cells, which may be useful as markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay following semi-automated RNA extraction from peripheral blood samples. Our approach includes a systematic search for candidate markers using digital differential display, search on UniGene colon EST libraries and analysis of published data on colon cancer gene expression. A final list included the following genes: bone morphogenetic protein 4 (BMP4), cyclin D (CycD), family with sequence similarity 3, member D (FAM3D), gastrin (GAS), glycoprotein A33 transmembrane (GPA33), glutathione peroxidase 2 gastrointestinal (GPX2), galactoside-binding, soluble, 4 (galectin 4) (LGALS4), non-SMC, structural maintenance of chromosomes, element 1 protein (NSE1), tumor-associated calcium signal transducer 1 (TACSTD1), telomerase reverse transcriptase (hTERT), trefoil factor 3 intestinal (TFF3), transmembrane 4 superfamily member 3 (TM4SF3), UDP glycosyltransferase 1 family, polypeptide A9 (UGT1A9), villin 1 (VIL1), and the novel gene FLJ20127. The mRNA expression of these genes was evaluated in a pool of 16 samples from subjects diagnosed with colon cancer and from 16 normal-controls. We observed expression in 13 of the 15 investigated genes from the blood samples of the vast majority of patients considered, but also in a certain percentage of the controls (from 14.3 to 100%). This finding confirms that the extreme sensitivity of RT-PCR is able to detect minimal amounts of mRNA expressed in a non tissue-specific manner ('illegitimate transcription'). On the contrary, NSE1 and GAS mRNAs were not detected either in patient or in control blood samples; however, they were abundantly expressed in normal and cancerous colon mucosa, encouraging further search for useful markers able to detect epithelial cells in peripheral blood.