Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant / Primera confirmación de importación y transmisión en España de la variante B.1.1.7 del SARS-CoV-2 recientemente identificada

Introduction: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. Results: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. Conclusion: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.(AU)

Introducción: Recientemente, ha surgido en Reino Unido una nueva variante de SARS-CoV-2, VOC202012/01, que origina el linaje B.1.1.7. Su rápida distribución en Reino Unido ha alertado a otros países a vigilar su presencia. Material y métodos: El rastreo inicial de la variante B.1.1.7 se basó en la ausencia de amplificación del gen S en el ensayo TaqPath, causado por la deleción 69/70. Todos los casos candidatos de corresponder a la variante B.1.1.7 con este criterio fueron posteriormente confirmados por secuenciación de genoma completo. Resultados: Describimos los primeros 3 casos importados de esta variante, desde Londres hasta Madrid, con la posterior transmisión domiciliaria de uno de estos casos a 3 familiares y, adicionalmente, los 2 primeros casos con la variante sin vínculo epidemiológico con Reino Unido. Uno de los casos requirió hospitalización. En todos los casos el criterio de no amplificación del gen S identificó con precisión la variante B.1.1.7, como demostró posteriormente la presencia de las 17 mutaciones marcadoras de este linaje. Conclusión: Las primeras identificaciones de la variante B.1.1.7 de SARS-CoV-2 indican un papel solapante de las introducciones independientes desde Reino Unido, con eventos de transmisión comunitaria, incluso desde los primeros momentos de la presencia de esta variante en nuestro país.(AU)

Role of molecular diagnostics in the clinical management of SARS-CoV-2 infection: advantages and drawbacks.

The diagnosis of SARS-CoV-2 is based on the use of nucleic acid amplification tests (NAAT), especially rRT-PCR. The latter also allows us to quickly identify variants of concern. However, its use in follow-up of patients and the correlation between Ct value and the viability of the virus is controversial.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Concurrent progressive multifocal leukoencephalopathy and central nervous system infiltration by multiple myeloma: A case report.

Progressive multifocal leukoencephalopathy rarely occurs in patients with multiple myeloma. Intracranial central nervous system invasion is also an uncommon event in multiple myeloma, occurring in less than 1% of cases. We describe herein an exceptional case of coexisting progressive multifocal leukoencephalopathy and intraparenchymal central nervous system myeloma infiltration. A 73-year-old woman with relapsed multiple myeloma was treated with 15 cycles of lenalidomide and dexamethasone, but therapy had to be stopped because of a hip fracture after a fall. During hospitalization, the patient developed progressive multifocal leukoencephalopathy caused by John Cunningham virus, and a prominent intra-parenchymal CD138-positive infiltrate was detected. VDJ rearrangements of the immunoglobulin heavy chain gene and the mutational profile of plasma cells in bone marrow at the time of diagnosis and in brain biopsy after progression were analyzed by next generation sequencing, showing genetic differences between medullary and extramedullary myeloma cells. The role of long-term treatment with lenalidomide and dexamethasone in the development progressive multifocal leukoencephalopathy or intraparenchymal central nervous system myeloma infiltration remains unknown. However, our results suggest that both events may have arisen as a consequence of treatment-related immunosuppression. Thus, an appropriate clinical approach compatible with the simultaneous treatment of progressive multifocal leukoencephalopathy and multiple myeloma should be developed.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Abstract Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Resumo Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 μg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer.

OBJECTIVES: Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS: Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS: Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS: Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 µg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Detection of Epstein-Barr virus DNAemia after lung transplantation and its potential relationship with the development of post-transplant complications.

BACKGROUND: Recent studies suggest that Epstein-Barr virus DNAemia (EBVd) may act as a surrogate marker of post-transplant immunosuppression. This hypothesis has not been tested so far in lung transplant (LT) recipients. METHODS: We included 63 patients undergoing lung transplantation at our center between October 2008 and May 2013. Whole blood EBVd was systematically assessed by real-time polymerase chain reaction assay on a quarterly basis. The occurrence of late complications (overall and opportunistic infection [OI] and chronic lung allograft dysfunction [CLAD]) was analyzed according to the detection of EBVd within the first 6 months post transplantation. RESULTS: Any EBVd was detected in 30 (47.6%) patients. Peak EBVd was higher in patients with late overall infection (2.23 vs. 1.73 log10 copies/mL; P = 0.026) and late OI (2.39 vs. 1.74 log10 copies/mL; P = 0.004). The areas under receiver operating characteristic curves for predicting both events were 0.806 and 0.871 respectively. The presence of an EBVd ≥2 log10 copies/mL during the first 6 months post transplantation was associated with a higher risk of late OI (adjusted hazard ratio [aHR] 7.92; 95% confidence interval [CI] 2.10-29.85; P = 0.002). Patients with detectable EBVd during the first 6 months also had lower CLAD-free survival (P = 0.035), although this association did not remain statistically significant in the multivariate analysis (aHR 1.26; 95% CI 0.87-5.29; P = 0.099). CONCLUSIONS: Although preliminary in nature, our results suggest that the detection of EBVd within the first 6 months after transplantation is associated with the subsequent occurrence of late OI in LT recipients.

Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major cause of long-term morbidity and mortality after heart transplantation (HTx), whose relationship with CMV infection is uncertain. This study evaluated the influence of CMV infection in the development of CAV. METHODS: We enrolled 166 consecutive HTx recipients who underwent their first transplant from January 1995 to July 2002. All patients received 14 days of intravenous ganciclovir and were prospectively monitored for CMV infection during the first year after HTx. CAV was diagnosed by coronary angiography performed at 1, 5, and 10 years after HTx, following the new criteria of the International Society for Heart and Lung Transplantation. We collected all variables potentially related with the development of CAV. Risk factors were studied using a complementary log-log model. RESULTS: After a median follow-up of 11 years (range, 1-17 years), 72 patients (43%) developed CAV (63.8% CAV(1), 15.2% CAV(2), 20.8% CAV(3)). Symptoms secondary to CAV were present in 32% of these patients, and 8% died because of it. In the regression multivariate analysis, independent variables associated with the development of CAV were donor age (hazard ratio [HR], 1.028; 95% confidence interval [CI], 1.002-1.053; p < 0.028), presence of cellular acute rejection ≥ 2R (HR, 1.764; 95% CI, 1.011-3.078; p < 0.0414), CMV infection (HR, 2.334; 95% CI, 1.043-5.225; p < 0.0354), and not having been treated with a calcium channel blocker (HR, 0.472; 95% CI, 0.275-0.811; p < 0.0055). CONCLUSIONS: Standardized angiographic criteria show CMV infection is associated with the development of CAV.

Everolimus-based immunosuppression therapy for BK virus nephropathy.

BACKGROUND: Mammalian target of rapamycin inhibitors (mTOR-i) have been proposed as possible immunosuppressants of choice in BK virus nephropathy (BKN) because of their antiviral capacity. On this basis, in 2007, our Service proposed a conversion to everolimus (EVE)-based therapy from calcineurin inhibitors with an anti-calcineurin-free therapy protocol in those patients diagnosed of BKN. METHODS: A prospective, single-center case series study was performed. Fifteen cases of BKN were diagnosed from 2007 to the end of 2010. According to our protocol, immunosuppressant treatment was modified in 9 of these patients with suspension of mycophenolate and conversion from tacrolimus to EVE. RESULTS: The renal function achieved by our patients after the transplantation was excellent. Mean serum creatinine (sCr) achieved was 1.16 ± 0.2 mg/dL. Evolution of the renal function after BKN diagnosis and conversion to mTOR-i was positive in all the patients. sCr on diagnosis was 1.85 ± 0.22 mg/dL, sCr at the point in time of conversion to EVE was 2 ± 0.21 mg/dL, and final sCr of the follow-up was 1.6 ± 0.39 mg/dL (P = .05). BK viremia became negative in 5 of our patients and decreased more than 95% in the remaining 4. None of the patients had an acute rejection episode after the change of immunosuppressant. CONCLUSIONS: Conversion to mTOR-i-based therapy could provide an added benefit in BKN and could be an effective strategy for the decrease of the viremia and increase of graft survival in selected patients.

Could polymerase chain reaction tests on conjunctival swabs be useful to diagnose herpetic keratitis?

INTRODUCTION: Diagnosis of HSV-1 keratitis (HK) is frequently based on clinical findings. Invasive specimens (corneal scrapings, biopsies) are required for microbiological diagnosis. Methods Corneal scrapings and conjunctival swabs were collected on patients with/without clinical suspicion of HK from 2007 to 2012.ResultsThe sensitivity, specificity, positive and negative predictive values for conjunctival swabs by PCR was 77.8, 92.1, 84.4 and 88.3, respectively. Discussion Conjunctival swabs by PCR may help in the diagnosis of HK, despite the limited sensitivity

INTRODUCCIÓN: El diagnóstico de queratitis herpética (QH) está basado normalmente en hallazgos clínicos. Para el diagnóstico microbiológico se requieren muestras invasivas (raspado corneal, biopsias).MÉTODOS: Raspados corneales y exudados conjuntivales fueron obtenidos de pacientes con/sin sospecha clínica de QH del ano˜ 2007 al 2012.RESULTADOS: La sensibilidad, la especificidad y los valores predictivos positivos y negativos para la PCR enexudados conjuntivales fueron 77,8, 92,1, 84,4 y 88,3, respectivamente. DISCUSIÓN: La PCR en exudados conjuntivales puede ayudar en el diagnóstico, a pesar de su limitada sensibilidad

Could polymerase chain reaction tests on conjunctival swabs be useful to diagnose herpetic keratitis?

INTRODUCTION: Diagnosis of HSV-1 keratitis (HK) is frequently based on clinical findings. Invasive specimens (corneal scrapings, biopsies) are required for microbiological diagnosis. METHODS: Corneal scrapings and conjunctival swabs were collected on patients with/without clinical suspicion of HK from 2007 to 2012. RESULTS: The sensitivity, specificity, positive and negative predictive values for conjunctival swabs by PCR was 77.8, 92.1, 84.4 and 88.3, respectively. DISCUSSION: Conjunctival swabs by PCR may help in the diagnosis of HK, despite the limited sensitivity.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86 percent of tumors (87 percent sensitivity and 80 percent specificity for predicting response), according to their response to chemotherapy (82 percent in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71 percent samples from the biological validation set were also correctly classified by both trios (72 percent sensitivity; 66 percent specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93 percent of samples from the technical validation group (95 percent sensitivity and 80 percent specificity; 86 percent accuracy by the cross-validation method) and 79 percent from the biological validation group (72 percent sensitivity and 100 percent specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancer patients.

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1 based trios, with NOTCH1 or NUP210. Both trios correctly separated 86% of tumors (87% sensitivity and 80% specificity for predicting response), according to their response to chemotherapy (82% in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71% samples from the biological validation set were also correctly classified by both trios (72% sensitivity; 66% specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93% of samples from the technical validation group (95% sensitivity and 80% specificity; 86% accuracy by the cross-validation method) and 79% from the biological validation group (72% sensitivity and 100% specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Human breast tumor slices: a model for identification of vitamin D regulated genes in the tumor microenvironment.

While many studies have addressed the direct effects of 1alpha,25(OH)2D3 on breast cancer (BC) cells, stromal-epithelial interactions, which are important for the tumor development, have been largely ignored. In addition, high concentrations of the hormone, which cannot be attained in vivo, have been used. Our aim was to establish a more physiological breast cancer model, represented by BC tissue slices, which maintain epithelial-mesenchymal interactions, cultured with a relatively low 1alpha,25(OH)2D3 concentration, in order to evaluate the vitamin D pathway. Freshly excised human BC samples were sliced and cultured in complete culture media containing vehicle, 0.5 nM or 100 nM 1alpha,25(OH)2D3 for 24 h. BC slices remained viable for at least 24 h, as evaluated by preserved tissue morphology in hematoxylin and eosin (HE) stained sections and bromodeoxyuridine (BrdU) incorporation by 10% of tumor cells. VDR mRNA expression was detected in all samples and CYP24A1 mRNA expression was induced by 1alpha,25(OH)2D3 in both concentrations (but mainly with 100 nM). Our results indicate that the vitamin D signaling pathway is functional in BC slices, a model which preserves stromal-epithelial interactions and mimics in vivo conditions.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas.

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow.

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.