CD133 expression in circulating tumor cells from breast cancer patients: potential role in resistance to chemotherapy.

CD133 has been associated with cell properties such as self renewal, migration and vasculogenic mimicry, potentially involved in generation of circulating tumor cells (CTCs). We characterized CD133 expression in CTCs of 98 nometastatic breast cancer (BC) patients. CTCs were isolated by immunomagnetic techniques using magnetic beads labeled with a multicytokeratin(CK)-specific antibody (CK3-11D5) and CTCs and CD133 detection through immunocytochemical methods. CK(+) /CD133(+) CTCs were identified in 65% of patients at baseline and 47.8% after systemic therapy (p = 0.53). Correlation of CD133 status in CTCs with classical clinicopathological characteristics and response to therapy was performed. Her2 not amplified and low Ki-67 index were positively correlated with presence of CK(+) /CD133(+) CTCs. Before any treatment, CK(+) /CD133(+) CTCs were more frequently isolated in patients with luminal BC subtype. No statistically significant differences were found between proportion of CK(+) /CD133(+) CTCs and BC subtypes after systemic therapy, implying a relative enrichment of CK(+) /CD133(+) CTCs in triple negative and HER2-amplified tumors. While CK(+) /CTCs decreases after chemotherapy when analyzing the whole population, CK(+) /CD133(+) CTCs were enriched in post-treatment samples in nonluminal BC subtypes. These findings suggest the potential role of CD133 as a promising marker of chemoresistance in nonluminal BC patients. Further prospective studies and extensive preclinical modeling will be needed to confirm whether CD133 is a marker of resistance to chemotherapy, and its role as a target for novel anticancer therapies targeting cancer stem cells and tumor vasculature.

Clinical validity of detecting K-ras mutations for the diagnosis of exocrine pancreatic cancer: a prospective study in a clinically-relevant spectrum of patients.

The diagnostic utility of detecting K-ras mutations for the diagnosis of exocrine pancreatic cancer (EPC) has not been properly studied, and few reports have analysed a clinically relevant spectrum of patients. The objective was to evaluate the clinical validity of detecting K-ras mutations in the diagnosis of EPC in a large sample of clinically relevant patients. We prospectively identified 374 patients in whom one of the following diagnoses was suspected at hospital admission: EPC, chronic pancreatitis, pancreatic cysts, and cancer of the extrahepatic biliary system. Mutations in the K-ras oncogene were analysed by PCR and artificial RFLP in 212 patients. The sensitivity and specificity of the K-ras mutational status for the diagnosis of EPC were 77.7% (95% CI: 69.2-84.8) and 78.0% (68.1-86.0), respectively. The diagnostic accuracy was hardly modified by sex and age. In patients with either mutated K-ras or CEA > 5 ng/ml, the sensitivity and specificity were 81.0% (72.9-87.6) and 62.6% (72.9-87.6), respectively. In patients with mutated K-ras and CEA > 5 ng/ml the sensitivity was markedly reduced. In comparisons with a variety of non-EPC patient groups sensitivity and specificity were both always greater than 75%. In this clinically relevant sample of patients the sensitivity and specificity of K-ras mutations were not sufficiently high for independent diagnostic use. However, it seems premature to rule out the utility of K-ras analysis in conjunction with other genetic and 'omics' technologies.

Food and nutrient intakes and K-ras mutations in exocrine pancreatic cancer.

BACKGROUND: No studies have investigated the relation between K-ras mutations and dietary factors in exocrine pancreatic cancer (EPC), and fewer than 10 studies have done so in other neoplasms. PATIENTS AND METHODS: Incident cases of EPC were prospectively identified, and interviewed face-to-face during hospital admission. Food and nutrient intakes were measured with a food frequency questionnaire. Logistic regression was used to compare EPC cases (n = 107) with and without K-ras mutations (case-case study). RESULTS: K-ras mutations were more common among daily consumers of milk and other dairy products than among non-daily consumers: the odds ratio adjusted by total energy, age, sex, smoking, alcohol and coffee consumption (ORa) was 5.1 (95% CI 1.1 to 24.5, p = 0.040). For all dairy products, including butter, the ORa for the medium and upper tertiles of intake were 5.4 and 11.6, respectively (p for trend = 0.023). The ORa for regular coffee drinkers further adjusted by dairy consumption was 4.7 (95% CI 1.1 to 20.7, p = 0.043). K-ras mutated cases reported a lower intake of vitamin E (ORa = 0.2, p for trend = 0.036), polyunsaturated fats and omega 3 fatty acids (ORa = 0.2; p for trend <0.03). CONCLUSIONS: Results support the hypothesis that in EPC exposure to specific dietary components or contaminants may influence the occurrence or persistence of K-ras mutations.

Mammographic extent of microcalcifications and oestrogen receptor expression affect preoperative breast carcinoma in situ size estimation.

BACKGROUND: The aim of our study was to establish which clinical, radiologic and pathologic factors could predict the risk of under- and overestimation of the breast ductal carcinoma in situ (DCIS) size when preoperatively measuring the maximum mammographic extent of microcalcifications (MEM). METHODS: We made a retrospective review of patients with a DCIS treated in our Breast Unit between May 2005 and May 2012. Clinical, pathologic and radiologic data were evaluated as possible predictive factors for over- or underestimation of DCIS size when measuring MEM. RESULTS: We obtained precise measurements of MEM in 82 patients (84 DCIS lesions). Maximum MEM measurement correctly estimated maximum pathology size in 57 lesions (68.7 %). Patients with a correctly estimated DCIS, with an underestimated DCIS and with an overestimated DCIS significantly differed in DCIS ER expression (p = 0.022) and in maximum MEM measurement (p = 0.000). Constructing two ROC curves, we found that a maximum MEM measurement ≥25 mm and ER expression ≥90 % were both discrimination points for overestimation and ER ≤ 45 % was a discrimination point for underestimation. Using these cutoff points, we defined four groups of patients with different risks of over- and underestimation. CONCLUSIONS: Risk of over- or underestimation of DCIS size through MEM measurement depends on DCIS ER expression and MEM itself. Identifying which patients are at a significant risk of over- or underestimation could help the breast surgeon when discussing the surgical options with the patient.

Biomarkers expression in benign breast diseases and risk of subsequent breast cancer: a case-control study.

Women with benign breast diseases (BBD) have a high risk of breast cancer. However, no biomarkers have been clearly established to predict cancer in these women. Our aim was to explore whether estrogen receptor (ER), progesterone receptor (PR), and Ki67 expression stratify risk of breast cancer in screened women with BBD. We conducted a nested case-control study. Women with breast cancer and prior BBDs (86 cases) were matched to women with prior BBDs who were free from breast cancer (172 controls). The matching factors were age at BBD diagnosis, type of BBD, and follow-up time since BBD diagnosis. ER, PR, and Ki67 expression were obtained from BBDs' specimens. Conditional logistic regression was used to estimate odds ratios (ORs), and 95% confidence intervals (CIs) of breast cancer risk according to ER, PR, and Ki67 expression. Women with >90% of ER expression had a higher risk of breast cancer (OR = 2.63; 95% CI: 1.26-5.51) than women with ≤70% of ER expression. Similarly, women with >80% of PR expression had a higher risk of breast cancer (OR = 2.22; 95% CI: 1.15-4.27) than women with ≤40% of PR expression. Women with proliferative disease and ≥1% of Ki67 expression had a nonsignificantly increased risk of breast cancer (OR = 1.16; 95% CI: 0.46-2.90) than women with <1% of Ki67 expression. A high expression of ER and PR in BBD is associated with an increased risk of subsequent breast cancer. In proliferative disease, high Ki67 expression may also have an increased risk. This information is helpful to better characterize BBD and is one more step toward personalizing the clinical management of these women.

The MYC oncogene in breast cancer progression: from benign epithelium to invasive carcinoma.

One hypothesis for breast cancer development suggests that breast carcinogenesis involves a progression of events leading from benign epithelium to hyperplasia (with or without atypia) to carcinoma in situ and then invasive carcinoma. The MYC gene (alias c-Myc) is a transcriptional regulator whose expression is strongly associated with cell proliferation and cell differentiation. The present study is a descriptive analysis of MYC status throughout the hypothesized stages of invasive ductal carcinoma progression. A tissue microarray (TMA) was constructed including representative selected areas (normal cells, hyperplasia, in situ carcinoma, and invasive carcinoma) from each of 15 patients. Fluorescence in situ hybridization (FISH) with the LSI c-MYC/CEN8/IgH probe was performed. Two cases displayed MYC amplification (13%), showing this amplification only in the invasive carcinoma zones selected. Five cases displayed polysomy of chromosome 8 (33%), detected only in ductal in situ and invasive zones selected. Benign lesions and normal adjacent cells were classified as normal. None of the hyperplasia specimens and normal specimens analyzed showed any alterations in MYC status or any aneusomies of chromosome 8. The presence of MYC amplification only in invasive cells suggests that the finding of MYC amplification could reflect an advanced tumor progression.

Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry.

INTRODUCTION: One of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC). METHODS: We used FISH on a series of 175 formalin-fixed paraffin-embedded breast carcinomas to detect ERBB2 amplification, using a dual-probe system for the simultaneous enumeration of the ERBB2 gene and the centromeric region of chromosome 17, as well as using IHC to detect overexpression. We analyzed clinical and pathological variables in a subgroup of patients with 2+ and 3+ IHC scores (147 patients), to describe any differences in clinicopathological characteristics between polysomic and non-polysomic cases with the use of the chi2 test. RESULTS: We found 13% of cases presenting polysomy, and three cases presented monosomy 17 (2%). According to the status of the ERBB2 gene, instances of polysomy 17 were more frequently observed in non-amplified cases than in FISH-amplified cases, suggesting that the mechanism for ERBB2 amplification is independent of polysomy 17. Polysomy 17 was detected in patients with 2+ and 3+ IHC scores. We found that nodal involvement was more frequent in polysomic than in non-polysomic cases (P = 0.046). CONCLUSIONS: The determination of the copy number of chromosome 17 should be incorporated into the assesment of ERBB2 status. It might also be helpful to differentiate a subgroup of breast cancer patients with polysomy of chromosome 17 and overexpression of ERBB2 protein that probably have genetic and clinical differences.

Exocrine pancreatic cancer: symptoms at presentation and their relation to tumour site and stage.

INTRODUCTION: The need to detect pancreatic cancer at earlier stages is undisputed. We recorded the signs and symptoms of patients presenting with exocrine pancreatic cancer and evaluated their association with clinical characteristics such as tumour site and disease stage. PATIENTS AND METHODS: All patients (n = 185) with exocrine pancreatic cancer newly diagnosed at five general hospitals in Eastern Spain were prospectively recruited over 5 years. Symptoms were elicited through personal interviews and signs were recorded by the attending physician on admission. RESULTS: At diagnosis, one third of tumours of the pancreas head were in stage I and another third in stage IV. None of the tumours of the body and tail were in stage I, and over 80% were in stage IV (p < 0.001) . At presentation, the most frequent symptoms were asthenia (86%), anorexia (85%), weight-loss (85%), abdominal pain (79%), and choluria (59%). Cholestatic symptoms were more common in tumours affecting only the pancreatic head (p < 0.001) . There was a clear trend toward more localized tumours with increasing numbers of cholestatic signs (p < 0.001) . Asthenia, anorexia and weight-loss were unrelated to stage. An increased symptom-to-diagnosis interval was associated with more advanced stage (p = 0.048). CONCLUSIONS: Proper attention to signs and symptoms, especially cholestasis, may help identify patients with pancreatic cancer at an earlier stage. Results also provide a current picture of the semiology of pancreatic cancer which could be of use in studies on the potential of proteomic tests in the early detection of this neoplasm.

Host cell-derived cardiomyocytes in sex-mismatch cardiac allografts.

BACKGROUND: Mesenchymal precursor cells are able to respond to tissue signals and differentiate into a phenotype characteristic of mature cells of that tissue. We sought to investigate whether adult human cardiomyocytes can be derived from recipient precursor cells in sex-mismatched cardiac allografts. METHODS: We studied four male patients who received hearts from female donors, and four female patients who received an allograft from a male donor. Four sex-matched transplant patients, two of each sex served as controls. Combined fluorescence in situ hybridization with probes specific for X- and Y-chromosomes and immunohistochemistry with alpha-actin was used to identify cardiac muscle cells 4 and 12 months after transplantation. Slides were examined with a fluorescence microscope to detect the presence of male cells with one X and one Y signal in the nucleus, and female cells containing two X signals. RESULTS: Mature cardiomyocytes from the host (1-2%) were found in five endomyocardial biopsy specimens at 4 months, and in three specimens at 12 months. In addition, recipient cells negative for cytoplasmic alpha-actin were also identified (1-21% per slide). The number of infiltrating recipient cells was not associated with the degree of rejection of the sample or with the number of prior rejection episodes. Echocardiographic evaluation showed no improvement in cardiac performance in hearts from patients with more than 10% chimeric recipient cells. CONCLUSIONS: Our data confirm the existence of mature cardiomyocytes derived from host cells, likely mesenchymal precursors, in the adult cardiac allograft in vivo.

Generalizing molecular results arising from incomplete biological samples: expected bias and unexpected findings.

PURPOSE: In molecular epidemiology, obtaining biological samples for all subjects targeted for study is frequently hampered by ethical, clinical, and logistic factors. The extent to which the incompleteness of biological samples could cause bias is rarely analyzed in depth. Here we report some expected bias and some unexpected findings during a study on mutations in the K-ras gene in exocrine pancreatic cancer (EPC). METHODS: In this case-case study, all patients registered with EPC between 1980 and 1990 at two general hospitals were retrospectively identified from the hospital tumor registries. Their clinical records were abstracted and paraffin-embedded samples retrieved from pathology records. DNA was amplified, and mutations in codon 12 of the K-ras gene were detected using the artificial RFLP technique. RESULTS: Results on the mutations (RM) were obtained for 51 of the 149 cases of EPC (34.2%). There were no significant differences on the availability of RM by age, gender, and tumor stage at diagnosis, but RM were over five times more likely to be available from one of the hospitals. Subjects with RM were more likely to have received a treatment with curative intent (OR = 11.56, 95% CL: 2.88-46.36). The existence of RM was positively associated with the availability of information on alcohol use and family history of cancer. Subjects with RM tended to belong to higher occupational groups and to smoke less than subjects without RM. Unexpectedly--given that in EPC K-ras mutations have consistently been found unrelated to age, gender, tumor stage, and other clinical factors-, cases with a K-ras mutation were more likely than wild-type cases to have information on tobacco and alcohol use (OR = 3.29, p = .21), medical history (OR = 4.46, p = .41), and family history of cancer (OR = 4.80, p = .01). The relationship between completeness of clinical records and K-ras mutations among cases with RM could not be accounted by age, gender, and occupational group. CONCLUSIONS: Simple tests of age and gender distributions among subjects with and without available clinical information and molecular results may not rule out selection and information bias. Studies using biologic specimens are even more in need than classic studies to explain clearly the process followed to include and exclude subjects. Additional caution is needed when generalizing molecular results arising from incomplete biological specimens.

Prediction of recurrence and nucleolar features in node-negative breast carcinoma, ductal type, grade II. An ultrastructural study.

One-third of patients with node-negative breast carcinoma develop a local recurrence that will decisively influence their outcome. We conducted this quantitative ultrastructural study to determine the value of nucleolar features in predicting recurrence of node-negative breast carcinomas. Forty-three node-negative infiltrating duct carcinomas were examined by means of electron microscopy. The number of cells with three or more nucleoli (N3), percentage of nucleoli contacting the nuclear membrane (NC), patient age, tumor size, grading, treatment, follow-up time, development of recurrence, and status were recorded. There were no differences in patient age, tumor grade, or modality of surgical treatment. Significant differences were found among patients with and without recurrence for N3 ( P=0.02), NC ( P=0.01), and tumor size ( P=0.01). For N3, the positive predictive value (PPV) was 64.3, and the negative predictive value (NPV) was 72.4 (sensitivity=52.9%; specificity=80.8%; P=0.04). For NC, the PPV was 62.5, and the NPV was 74.1 (sensitivity=58.8%; specificity=76.9%; P=0.02). For tumor size, the PPV was 66.7, and the NPV was 64.9 (sensitivity=23.5%; specificity=92.3%; P not significant). Using logistic regression analysis, independent predictive value was shown for N3, NC, and tumor size. The number of nucleoli per cell and the number of cells with nucleoli in contact with the nuclear membrane have independent predictive value for the development of recurrence in lymph node-negative infiltrating duct carcinoma of the breast. Although electron microscopy is a highly accurate method, improving the precision of light microscopic techniques would allow more universal assessment of these nucleolar features.

Are ER+PR+ and ER+PR- breast tumors genetically different? A CGH array study.

The estrogen receptor (ER) is a well-known predictor of breast cancer response to endocrine therapy. ER+ progesterone receptor (PR)- breast tumors have a poorer response to endocrine therapy and a more aggressive phenotype than ER+PR+ tumors. A comparative genomic hybridization array technique was used to examine 25 ER+PR+ and 23 ER+PR- tumors. Tissue microarrays composed of 50 ER+PR+ and 50 ER+PR- tumors were developed to validate the comparative genomic hybridization array results. The genes of interest were analyzed by fluorescence in situ hybridization. The ER+PR- group had a slightly different genomic profile when compared with ER+PR+ tumors. Chromosomes 17 and 20 contained the most overlapping gains, and chromosomes 3, 8, 9, 14, 17, 21, and 22 contained the most overlapping losses when compared with the ER+PR+ group. The gained regions, 17q23.2-q23.3 and 20q13.12, and the lost regions, 3p21.32-p12.3, 9pter-p13.2, 17pter-p12, and 21pter-q21.1, occurred at different alteration frequencies and were statistically significant in the ER+PR- tumors compared with the ER+PR+ tumors. ER+PR- breast tumors have a different genomic profile compared with ER+PR+ tumors. Differentially lost regions in the ER+PR- group included genes with tumor suppressor functions and genes involved in apoptosis, mitosis, angiogenesis, and cell spreading. Differentially gained regions included genes such as MAP3K3, RPS6KB1, and ZNF217. Amplification of these genes could contribute to resistance to apoptosis, increased activation of the PI3K/Akt/mTOR pathway, and the loss of PR in at least some ER+PR- tumors.

Does polysomy of chromosome 17 have a role in ERBB2 and topoisomerase IIalpha expression? Gene, mRNA and protein expression: a comprehensive analysis.

OBJECTIVES: ERBB2 is an oncogene with prognostic and predictive value. Topoisomerase IIalpha is an enzyme encoding close to the ERBB2 oncogene, that represents a molecular target for anthracyclines. An indirect mechanism of increasing ERBB2 and topoisomerase IIalpha gene copy number is chromosome 17 polysomy. The aim of the present study was to clarify the implication of polysomy 17 in ERBB2 and topoisomerase IIalpha expression. In addition, we assessed the relation of ERBB2 and topoisomerase IIalpha gene dosage to mRNA and protein levels. METHODS: We selected 83 cases diagnosed as invasive breast cancer. We analysed ERBB2 and topoisomerase IIalpha genes, mRNA and protein by fluorescence in situ hybridisation, real-time reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: We observed a progressive increase in mRNA expression from 0+ to 3+ and also a significant difference in the ERBB2 RNA levels between normal and amplified cases. We found that polysomy of chromosome 17 does not affect the ERBB2 expression and that topoisomerase IIalpha mRNA expression is not related to gene status. CONCLUSIONS: Our results demonstrate that polysomy of chromosome 17 is not related to ERBB2 expression. Thereby, it is important to use centromeric probes to clearly discriminate between true ERBB2 gene amplification and polysomy of chromosome 17.

Tissue plasminogen activator induces pancreatic cancer cell proliferation by a non-catalytic mechanism that requires extracellular signal-regulated kinase 1/2 activation through epidermal growth factor receptor and annexin A2.

Tissue plasminogen activator (tPA) is overexpressed in pancreatic ductal carcinoma and is involved in tumor progression. This effect is probably mediated through the activation of angiogenesis, cell invasion, and cell proliferation. Previous studies support the notion that the effects of tPA on cell invasion require its proteolytic activity. Here, we report the molecular mechanism responsible for the proliferative effects of tPA on pancreatic tumor cells. tPA activates the extracellular signal-regulated kinase 1/2 signaling pathway in a manner that is independent of its catalytic activity. We also show that at least two membrane receptors, epidermal growth factor receptor and annexin A2, which are overexpressed in pancreatic cancer, are involved in the transduction of tPA signaling in pancreatic tumors. This observation suggests the establishment of an amplification loop in tumor cell proliferation. Double immunofluorescence experiments showed co-localization of tPA/epidermal growth factor receptor and tPA/annexin A2 in pancreas cancer cells. These results add novel insights into the non-catalytic functions of tPA in cancer and the molecular mechanisms behind the effects of this protease on cell proliferation, including a role for epidermal growth factor receptor.

Lifetime history of tobacco consumption and K-ras mutations in exocrine pancreatic cancer.

OBJECTIVES: We analyzed the relation between mutations in codon 12 of the K-ras oncogene and lifetime consumption of tobacco in patients with exocrine pancreatic cancer (EPC). METHODS: Incident cases of EPC were prospectively identified and interviewed during hospital admission about smoking and other factors. Exact logistic regression was used to compare EPC cases (N = 107) with and without K-ras mutations (case-case study). RESULTS: Mutated cases were nonsignificantly less likely to have been smokers than wild-type cases: the odds ratio adjusted by age and sex was 0.54 (95% confidence interval, 0.10-2.69; P = 0.613). With respect to never smokers, adjusted odds ratios for former and current smokers were 0.79 and 0.36, respectively (P = 0.193). Pack-years smoked, years of smoking, and cigarettes smoked per year also tended to be higher in nonmutated than in mutated cases. Neither age at onset of smoking nor the time between quitting and diagnosis were associated with K-ras. CONCLUSIONS: Tobacco does not play a major part in the acquisition of K-ras mutations in the pancreatic epithelium. Although both smoking and K-ras mutations have important roles in the etiopathogenesis of EPC, the 2 processes may act independently.

Oxidative stress and respiratory muscle dysfunction in severe chronic obstructive pulmonary disease.

RATIONALE: Oxidative stress is involved in the skeletal muscle dysfunction observed in patients with severe chronic obstructive pulmonary disease (COPD). We hypothesized that the diaphragms of such patients might generate greater levels of oxidants than those neutralized by antioxidants. OBJECTIVES: To assess the levels of both oxidative and nitrosative stress and different antioxidants in the diaphragms of those patients, and to analyze potential relationships with lung and respiratory muscle dysfunctions. METHODS AND MEASUREMENTS: We conducted a case-control study in which reactive carbonyl groups, hydroxynonenal-protein adducts, antioxidant enzyme levels, nitric oxide synthases, and 3-nitrotyrosine formation were detected using immunoblotting and immunhistochemistry in diaphragm specimens (thoracotomy) obtained from six patients with severe COPD, six patients with moderate COPD, and seven control subjects. MAIN RESULTS: Diaphragms of patients with severe COPD showed both higher protein carbonyl groups and hydroxynonenal-protein adducts than control subjects. When only considering patients with COPD, negative correlations were found between carbonyl groups and airway obstruction, and between hydroxynonenal-protein adducts and respiratory muscle strength. Although diaphragmatic neuronal nitric oxide synthase did not differ among the three groups and no inducible nitric oxide synthase was detected in any muscle, muscle endothelial nitric oxide synthase was lower in patients with severe COPD than in control subjects. Muscle nitrotyrosine levels were similar in both patients with severe COPD and control subjects. CONCLUSIONS: This study shows that oxidative stress rather than nitric oxide is likely to be involved in the respiratory muscle dysfunction in severe COPD.