Urinary microbiota is associated to clinicopathological features in benign prostatic hyperplasia.

BACKGROUND: The urinary microbiota of patients with benign prostatic hyperplasia (BPH) has been associated with lower urinary tract symptoms (LUTS), however, little is known about urinary microbiota correlations with clinicopathological parameters associated with BPH. Here, we investigate associations between the urinary microbiota and clinical parameters of patients with BPH undergoing surgery. METHODS: Forty-one patients with BPH undergoing surgery were recruited from two medical centers. Catheterized urine specimens were collected and the microbiota was characterized by 16S rRNA gene sequencing. Patients were segregated into two groups according to each clinical parameter and differences in urinary microbiota diversity and composition were evaluated. RESULTS: Higher prostate weight and prostate-specific antigen (PSA) levels were associated with higher alpha diversity in the urinary microbiota of BPH patients. At the specific microbe level, we found that the greater the prostatic weight, the lower the relative abundance of Streptococcus, while the greater the PSA levels, the higher the abundance of Lactobacillus. Treatment with 5-α-reductase inhibitor was associated with overall urinary microbiota composition, in part due to a higher abundance of Corynebacterium and Anaerococcus in this group. CONCLUSIONS: We demonstrated that the urinary microbiota of BPH patients is associated with clinicopathological features, paving the way for larger studies in which causality between urinary microbiota and BPH can be appropriately explored.

Suspected Germline TP53 Variants and Clonal Hematopoiesis of Indeterminate Potential: Lessons Learned From a Molecular Tumor Board.

OBJECTIVE: Li-Fraumeni syndrome (LFS) is a pan-cancer predisposition syndrome caused by germline pathogenic variants in the gene TP53. The interpretation of TP53 variants in clinical scenarios outside the classic LFS criteria may be challenging. Here, we report a patient affected by 2 primary cancers at later ages, who harbored a likely pathogenic TP53 at low allele frequency detected in a blood sample. METHODS: The Molecular Tumor Board committee at our institution revisited the case of a patient who was enrolled in a research protocol for the investigation of genetic conditions associated with neuroendocrine tumors. Clinical, familial, and molecular data were reviewed. The patient received germline testing using a next generation sequencing multi-gene panel and was incidentally found to harbor a TP53 likely pathogenic variant, with 22% of variant allele fraction. Additional samples, including a second blood sample, oral swab, and saliva, were collected for DNA analysis. A new TP53 sequencing round was performed with the attempt to distinguish between a true constitutional germline variant and a somatically acquired variant due to aberrant clonal expansion of bone marrow precursors. RESULTS: Patient's personal and familial history of cancer did not meet classic nor Chompret LFS criteria. Environmental risk factors for cancer were identified, such as alcohol abuse and tobacco exposure. The TP53 variant initially found in the next-generation sequencing was confirmed by Sanger sequencing in the previous DNA sample extracted from blood for the first analysis and in a second blood sample collected 6 years later. The TP53 variant was not detected in the DNA extracted from the oral swab and saliva samples. CONCLUSION: Considering the low TP53 variant allele fraction in blood, absence of variant detection in oral swab and saliva samples, the lack of LFS clinical criteria, and history of exposure to environmental risk factors for cancer, the main hypothesis for this case was aberrant clonal expansion due to clonal hematopoiesis. Oncologists should interpret TP53 findings during germline testing with caution.

Monoallelic deleterious MUTYH germline variants as a driver for tumorigenesis.

MUTYH encodes a glycosylase involved in the base excision repair of DNA. Biallelic pathogenic germline variants in MUTYH cause an autosomal recessive condition known as MUTYH-associated adenomatous polyposis and consequently increase the risk of colorectal cancer. However, reports of increased cancer risk in individuals carrying only one defective MUTYH allele are controversial and based on studies involving few individuals. Here, we describe a comprehensive investigation of monoallelic pathogenic MUTYH germline variants in 10,389 cancer patients across 33 different tumour types and 117,000 healthy individuals. Our results indicate that monoallelic pathogenic MUTYH germline variants can lead to tumorigenesis through a mechanism of somatic loss of heterozygosity of the functional MUTYH allele in the tumour. We confirmed that the frequency of monoallelic pathogenic MUTYH germline variants is higher in individuals with cancer than in the general population, although this frequency is not homogeneous among tumour types. We also demonstrated that the MUTYH mutational signature is present only in tumours with loss of the functional allele and found that the characteristic MUTYH base substitution (C>A) increases stop-codon generation. We identified key genes that are affected during tumorigenesis. In conclusion, we propose that carriers of the monoallelic pathogenic MUTYH germline variant are at a higher risk of developing tumours, especially those with frequent loss of heterozygosity events, such as adrenal adenocarcinoma, although the overall risk is still low. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Oral shedding of herpesviruses and clinical outcomes in hematopoietic stem cell transplant patients.

OBJECTIVES: To characterize the oral shedding of herpes viruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and investigate its relationship with clinical outcomes. MATERIALS AND METHODS: Polymerase chain reaction and enzymatic digestion were performed to identify the oral shedding of the members of the Herpesviridae family in 31 patients. The samples were collected from the oral cavity at five timestamps. RESULTS: The presence of each herpesvirus in the oral cavity was observed in 3.2%, 12.9%, 19.3%, 32.2%, 54.8% and 93.5% patients for human herpesvirus (HHV)-6A, herpes simplex virus-1, HHV-6B, cytomegalovirus (CMV), Epstein-Barr virus (EBV) and HHV-7, respectively. Oral shedding of herpes virus was not uncommon after alloHSCT. There was a statistically significant association between the EBV and CMV oral shedding at C1 and the cumulative incidence of acute graft-versus-host disease (aGVHD). The results suggested that the presence of HSV-1 at C2 was related to a relapse. The HHV-7 oral shedding at C2 suggests a possible link between relapse, progression-free survival and overall survival of the patients. CONCLUSIONS: Patients who developed aGVHD showed higher CMV and EBV shedding in the oral cavity at aplasia, suggesting modifications to the pattern of immune cell response and inflammatory microenvironment.

Unusual gingival actinomycosis post allogeneic hematopoietic stem-cell transplant: case report.

BACKGROUND: Allogeneic hematopoietic stem cell transplant (allo-HSCT) is used to treat several hematological diseases, but immunosuppression during allo-HSCT facilitates opportunistic microbial growth in tissues, such as actinomycosis. An effective diagnosis of opportunistic diseases is essential for correct management of the disease and preservation of the immunosuppressed patient's life. CASE DESCRIPTION: A 57-year-old female patient was diagnosed with extranodal nasal type NK/T cell lymphoma and underwent curative treatment with allo-HSCT. Twenty-one days after the last clinical follow-up, the patient presented a necrotizing lesion in the papilla region between the first and second molars of the second quadrant. Histopathological analysis showed the presence of a bacterial cluster consistent with Actinomyces infection, and a dense lymphoid infiltrate was also observed. Immunohistochemistry for CD20, CD3, and CD56 was performed to exclude the possibility of the recurrence of extranodal NK/T cell lymphoma. Oral microbiota profiling showed a huge increase in the abundance of Actinomyces bacteria in the subgingival region three weeks prior to appearance of the lesion. CONCLUSIONS: Opportunistic infections with an unusual clinical appearance are confounding factors in therapeutic decision-making. We present for the first time a case of actinomycosis in the gingival papilla region following allo-HSCT. We also highlight how microbiota profiling through next-generation sequencing could be used to anticipate bacterial infection diagnosis.

A longitudinal study of convalescent plasma (CCP) donors and correlation of ABO group, initial neutralizing antibodies (nAb), and body mass index (BMI) with nAb and anti-nucleocapsid (NP) SARS-CoV-2 antibody kinetics: Proposals for better quality of CCP collections.

INTRODUCTION: Little is known about the neutralizing (nAb) and binding antibody kinetics in COVID-19 convalescent plasma donors, especially during the first 100 days after disease onset. MATERIALS AND METHODS: A cohort of previously RT-PCR positive (detected by nasopharyngeal swab during the acute phase), male convalescent patients, all with mild symptoms, were enrolled in serial blood sample collection for a longitudinal nAb titers and anti-nucleocapsid (NP) antibodies (IgM, IgG and IgA) evaluation. NAbs were detected by a cytopathic effect-based virus neutralization test (CPE-based VNT), carried out with SARS-CoV-2 (GenBank: MT350282). RESULTS: A total of 78 male volunteers provided 316 samples, spanning a total of 4820 days of study. Although only 25% of donors kept nAb titers ≥160 within 100 days after the onset of disease, there was >75% probability of sustaining nAb titers ≥160 in volunteers whose initial nAb titer was ≥1280, weight ≥ 90 kg or obese, according to their body mass index (BMI), as evidenced by Kaplan-Meier analysis and Cox hazard regression (all p < .02). There was no correlation between the ABO group, ABO antibody titers and persistent high nAb titers. High IgG anti-NP (S/CO ≥5.0) is a good surrogate for detecting nAb ≥ 160, defined by the ROC curve (sensitivity = 90.5%; CI95%: 84.5%-94.7%). CONCLUSION: Selection of CCP donors for multiple collections based on initial high nAb titers (≥1280) or BMI ≥ 30 kg/m2 provides a simple strategy to achieve higher quality in CCP programs. High IgG anti-NP levels can also be used as surrogate markers for high nAb screening.

COVID-19 convalescent plasma cohort study: Evaluation of the association between both donor and recipient neutralizing antibody titers and patient outcomes.

BACKGROUND: Current evidence regarding COVID-19 convalescent plasma (CCP) transfusion practices is limited and heterogeneous. We aimed to determine the impact of the use of CCP transfusion in patients with previous circulating neutralizing antibodies (nAbs) in COVID-19. METHODS: Prospective cohort including 102 patients with COVID-19 transfused with ABO compatible CCP on days 0-2 after enrollment. Clinical status of patients was assessed using the adapted World Health Organization (WHO) ordinal scale on days 0, 5, and 14. The nAbs titration was performed using the cytopathic effect-based virus neutralization test with SARS-CoV-2 (GenBank MT126808.1). The primary outcome was clinical improvement on day 14, defined as a reduction of at least two points on the adapted WHO ordinal scale. Secondary outcomes were the number of intensive care unit (ICU)-free days and the number of invasive mechanical ventilation-free days. RESULTS: Both nAbs of CCP units transfused (p < 0.001) and nAbs of patients before CCP transfusions (p = 0.028) were associated with clinical improvements by day 14. No significant associations between nAbs of patients or CCP units transfused were observed in the number of ICU or mechanical ventilation-free days. Administration of CCP units after 10 days of symptom onset resulted in a decrease in ICU-free days (p < 0.001) and mechanical ventilation-free days (p < 0.001). CONCLUSION: Transfusion of high titer nAbs CCP units may be a determinant in clinical strategies against COVID-19. We consider these data as useful parameters to guide future CCP transfusion practices.

Screening for SARS-CoV-2 antibodies in convalescent plasma in Brazil: Preliminary lessons from a voluntary convalescent donor program.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) collection began in two Brazilian hospitals for treatment of severe/critical patients. METHODS AND MATERIALS: Mild/moderate COVID-19 convalescents were selected as CCP donors after reverse transcription polymerase chain reaction (RT-PCR) confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and absence of symptoms for ≥14 days plus (a) age (18-60 years), body weight greater than 55 kg; (b) immunohematological studies; (c) no infectious markers of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T-lymphotropic virus-1/2, Chagas and syphilis infection; (d) no HLA antibodies (multiparous); (e) second RT-PCR (nasopharyngeal swab and/or blood) negativity; (f) virus neutralization test (cytopathic effect-based virus neutralization test neutralizing antibody) and anti-nucleocapsid protein SARS-CoV-2 IgM, IgG, and IgA enzyme-linked immunosorbent assays. RESULTS: Among 271 donors (41 females, 230 males), 250 presented with neutralizing antibodies. Final RT-PCR was negative on swab (77.0%) or blood (88.4%; P = .46). Final definition of RT-PCR was only defined at more than 28 days after full recovery in 59 of 174 (33.9%) RT-PCR -ve, and 25/69 RT-PCR +ve (36.2%; 13 between 35 and 48 days). Neutralizing antibody titers of 160 or greater were found in 63.6%. Correlation between IgG signal/cutoff of 5.0 or greater and neutralizing antibody of 160 or greater was 82.4%. Combination of final RT-PCR -ve with neutralizing antibody ≥160 was 41.3% (112/271). Serial plasma collection showed decline in neutralizing antibody titers and IgA levels (P < .05), probably denoting a "golden period" for CCP collection (≤28 days after joining the program); IgA might have an important role as neutralizing antibody. Donor's weight, days between disease onset and serial plasma collection, and IgG and IgM levels are important predictors for neutralizing antibody titer. CONCLUSIONS: RT-PCR +ve cases are still detected in 36.2% within 28 to 48 days after recovery. High anti-nucleocapsid protein IgG levels may be used as a surrogate marker to neutralizing antibody.

Metformin as an Alternative Radiosensitizing Agent to 5-Fluorouracil During Neoadjuvant Treatment for Rectal Cancer.

BACKGROUND: Neoadjuvant chemoradiation for locally advanced rectal cancer combining 5-fluorouracil with radiation increases tumor regression compared with radiation alone. However, it occurs at the cost of significant treatment-related toxicity. Patients with rectal cancer using metformin have been associated with improved response to radiotherapy. OBJECTIVE: The purpose of this study was to evaluate the radiosensitizing effects of metformin in vitro and in vivo and compare it with a standard combination of radiation/5-fluorouracil. DESIGN: Colorectal cancer cell lines SW480, HT29, and HCT116 were used as models. Cell viability was compared under treatments with radiation, radiation/5-fluorouracil, metformin, radiation/metformin, and radiation/5-fluorouracil/metformin. Nude mice were injected subcutaneously with SW480 cells and treated for 1 week with radiation/5-fluorouracil, metformin, radiation/metformin, or radiation/5-fluorouracil/metformin. Tumor volume was evaluated for 4 weeks after treatment completion. The phosphorylation status of key proteins of the PI3K/Akt/mTOR pathway was determined by immunoblots. SETTINGS: This was an experimental study conducted in vitro and in vivo. PATIENTS: Animal models/cell lines were used. MAIN OUTCOME MEASURES: The end point was to investigate how metformin compares with 5-fluorouracil as a radiosensitizer. RESULTS: All cell lines significantly decreased cell viability after treatment with radiation/metformin when compared with radiation alone. Radiation/metformin was superior to radiation/5-fluorouracil in SW480 (37% vs 74%; p < 0.001). In HT29 and in HCT116, radiation/metformin was inferior to radiation/5-fluorouracil (40.0% vs 13.8%, p < 0.001 and 40.0% vs 7.0%, p < 0.001), mainly because of increased 5-fluorouracil toxicity (≤20% of cell viability). In vivo assays indicated that radiation/metformin treatment was comparable with radiation/5-fluorouracil (557 vs 398 mm; p > 0.05) and that the addition of metformin to the standard radiation/5-fluorouracil did not improve tumor response (349 mm; p > 0.05). Metformin exerted strong PI3K/Akt/mTOR pathway inactivation effects after 24-hour exposure (increasing pAMPK, p < 0.01; decreasing pAkt, p < 0.01; and pS6, p <0.05). LIMITATIONS: In vitro and in vivo chemoradiation regimens cannot be directly translated to human delivery methods. CONCLUSIONS: Metformin enhances tumor response to radiation in vitro and in vivo. Metformin is an attractive alternative radiosensitizing agent to be considered in future studies/trials. See Video Abstract at http://links.lww.com/DCR/B219. LA METFORMINA COMO AGENTE RADIOSENSIBILIZADOR ALTERNATIVO A 5FU DURANTE EL TRATAMIENTO NEOADYUVANTE PARA CÁNCER DE RECTO: La quimiorradiación neoadyuvante para el cáncer de recto localmente avanzado que combina 5FU con radiación aumenta la regresión tumoral en comparación con la radiación sola. Sin embargo, se produce a costa de una toxicidad significativa relacionada con el tratamiento. Los pacientes con cáncer de recto que usan metformina se han asociado con una mejor respuesta a la radioterapia.Evaluar los efectos radiosensibilizantes de metformina in vitro e in vivo y compararlo con la combinación estándar de radiación / 5FU.Se usaron como modelos las líneas celulares de cáncer colorrectal SW480, HT29 y HCT116. La viabilidad celular se comparó en tratamientos con radiación, radiación / 5FU, metformina, radiación / metformina y radiación / 5FU / metformina. A los ratones desnudos se les inyectó por vía subcutánea células SW480 y fueron tratados durante una semana con radiación / 5FU, metformina, radiación / metformina o radiación / 5FU / metformina. El volumen tumoral se evaluó durante 4 semanas después de la finalización del tratamiento. El estado de fosforilación de las proteínas clave de la vía PI3K / Akt / mTOR se determinó mediante inmunotransferencias.Estudio experimental in vitro e in vivo.Modelo animal / líneas celulares.El punto final fue investigar cómo la metformina se compara con 5FU como un radiosensibilizador.Todas las líneas celulares disminuyeron significativamente la viabilidad celular después del tratamiento con radiación / metformina en comparación con la radiación sola. La radiación / metformina fue superior a la radiación / 5FU en SW480 (37% frente a 74%; p <0,001). En el HT29 y el HCT116 la radiación / metformina fue inferior a la radiación / 5FU (40% vs 13.8%, p <0.001 y 40% vs 7%, p <0.001; respectivamente), debido principalmente al aumento de la toxicidad de 5FU (≤20% de la célula viabilidad). Los ensayos in vivo indicaron que el tratamiento con radiación / metformina era comparable a la radiación / 5FU (557 vs 398 mm, p > 0.05), y que la adición de metformina a la radiación estándar / 5FU no mejoró la respuesta tumoral (349 mm, p > 0.05). La metformina ejerció fuertes efectos de inactivación de la vía PI3K / Akt / mTOR después de 24 horas de exposición (aumentando pAMPK p < 0.01, disminuyendo pAkt, p < 0.01; y pS6, p < 0.05).Los regímenes de CRT in vitro e in vivo no se pueden traducir directamente a los métodos de entrega en humanos.La metformina mejora la respuesta tumoral a la radiación in vitro e in vivo. La metformina es un agente alternativo de radiosensibilización atractivo para ser considerado en futuros estudios / ensayos. Consulte Video Resumen en http://links.lww.com/DCR/B219. (Traducción-Dr Gonzalo Hagerman).

Prediction of Poor Response to Neoadjuvant Chemoradiation in Patients With Rectal Cancer Using a DNA Repair Deregulation Score: Picking the Losers Instead of the Winners.

BACKGROUND: Patients with rectal cancer may undergo neoadjuvant chemoradiation even in early stages in an attempt to achieve complete clinical response and undergo organ preservation. However, prediction of tumor response is unavailable. In this setting, accurate identification of poor responders could spare patients with early stage disease from potentially unnecessary chemoradiation. OBJECTIVE: This study focused on development/test of a score based on DNA repair gene expression to predict response to neoadjuvant chemoradiation in patients with rectal cancer. DESIGN: Pretreatment biopsy samples from patients with rectal cancer undergoing neoadjuvant chemoradiation were collected and underwent gene expression analysis using RNA-Seq (test cohort). A score was constructed using 8 differentially expressed DNA repair genes between good (complete clinical) and poor responders (incomplete clinical) to treatment. The score was validated in 2 independent cohorts of patients undergoing similar treatment strategies and using quantitative polymerase chain reaction and microarray gene expression data. SETTINGS: This was a retrospective analysis of gene expression data from 3 independent institutions. PATIENTS: Patients with rectal cancer undergoing neoadjuvant chemoradiation (50.4-54.0 Gy and 5-fluorouracil-based chemotherapy) were eligible. Patients with complete clinical response, complete pathological response, or ≤10% residual cancer cells were considered good responders. Patients with >10% residual cancer cells were considered poor responders. The test cohort included 25 patients (16 poor responders). Validation cohort 1 included 28 patients (18 poor responders), and validation cohort 2 included 46 patients (22 poor responders). MAIN OUTCOMES MEASURES: Response was correlated with the DNA repair score calculated using the expression levels of 8 DNA repair genes. DNA repair score sensitivity, specificity, and positive and negative predictive values were determined in test and validation cohorts. RESULTS: Poor responders had significantly lower DNA repair scores when compared with good responders across all 3 cohorts, regardless of the gene expression platform used. A low score correctly predicted poor response in 93%, 90%, and 71% in test, validation 1, and validation 2 cohorts. LIMITATIONS: This study was limited by its small sample size, different gene expression platforms, and treatment regimens across different cohorts used. CONCLUSIONS: A DNA repair gene score may predict patients likely to have poor response to chemoradiation. This score may be a relevant tool to be investigated in future studies focused on chemoradiation used in the context of organ preservation. See Video Abstract at http://links.lww.com/DCR/B104. PREDICCIÓN DE RESPUESTA DEFICIENTE A LA RADIO-QUIMIOTERAPIA NEOADYUVANTE EN PACIENTES CON CÁNCER RECTAL UTILIZANDO UNA PUNTUACIÓN DE DESREGULACIÓN DE REPARACIÓN DE ADN: ESCOGER LOS PERDEDORES EN LUGAR DE LOS GANADORES: Los pacientes con cáncer rectal pueden someterse a radio-quimioterapia neoadyuvante incluso en estadios tempranos en el intento por lograr una respuesta clínica completa y permitir una preservación de órgano. Sin embargo, aun no existen herramientas disponible para la prediccion de la respuesta tumoral al tratamiento. En este contexto, la identificación precisa de los tumores con mala respuesta al tratamiento podría evitar que los pacientes con enfermedad en estadio temprano sean sometidos a radio-quimioterapia potencialmente innecesaria.Desarrollo / testeo de una puntuación basada en la expresión genes reparadores del ADN para predecir la respuesta a la nCRT en pacientes con cáncer rectal.Se recogieron muestras de biopsia de pre-tratamiento de pacientes con cáncer rectal sometidos a radio-quimioterapia neoadyuvante y se les realizó un análisis de expresión génica utilizando RNAseq (cohorte de prueba). Se construyó una puntuación utilizando 8 genes de reparación de ADN expresados diferencialmente entre buenos (respuesta clinica completa) y pobres respondedores (respuesta clinica incompleta) al tratamiento. La puntuación se validó en 2 cohortes independientes de pacientes sometidos a estrategias de tratamiento similares y utilizando qPCR y datos de expresión de genes en chips ADN (biotecnología -microarrays).Análisis retrospectivo de los datos de expresión génica de 3 instituciones independientes.Fueron incluidos aquellos pacientes con cáncer rectal sometidos a radio-quimioterapia neoadyuvante (50,4-54 Gy y quimioterapia basada en 5FU). Los pacientes con respuesta clínica completa, respuesta patológica completa o ≤10% de células cancerosas residuales se consideraron buenos respondedores. Los pacientes con> 10% de células cancerosas residuales se consideraron de respuesta deficiente. La cohorte de prueba incluyó a 25 pacientes (16 respondedores pobres). La cohorte de validación n. ° 1 incluyó a 28 pacientes (18 respondedores pobres) y la cohorte de validación n. ° 2 incluyó a 46 pacientes (22 respondedores pobres).La respuesta se correlacionó con la puntuación de reparación de ADN calculada utilizando los niveles de expresión de 8 genes de reparación de ADN. La sensibilidad del puntaje de reparación del ADN, la especificidad, los valores predictivos positivos y negativos se determinaron en las cohortes de prueba y validación.Los malos respondedores tuvieron puntuaciones de reparación de ADN significativamente más bajas en comparación con los buenos respondedores en las 3 cohortes, independientemente de la plataforma de expresión génica utilizada. Una puntuación baja predijo correctamente una respuesta pobre en el 93%, 90% y 71% en las cohortes de prueba, validación n. ° 1 y validación n. ° 2, respectivamente.Pequeño tamaño de la muestra, diferentes plataformas de expresión génica y regímenes de tratamiento en diferentes cohortes utilizadas.La puntuacion basada en genes de reparación del ADN puede predecir los pacientes con respuesta pobre a la radio-quimioterapia. Esta puntuación puede ser una herramienta relevante para investigar en futuros estudios centrados en la radio-quimioterapia utilizada en el contexto de la preservación de órganos. Consulte Video Resumen en http://links.lww.com/DCR/B104. (Traducción-Dr. Xavier Delgadillo and Dr. Laura Melina Fernandez).

Herpesviruses in the oral cavity of patients subjected to allogeneic hematopoietic stem cell transplantation and its relationship with oral mucositis.

OBJECTIVES: This study was performed to characterise oral shedding of herpesviruses in patients who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) and to investigate its relationship with oral mucositis (OM). MATERIALS AND METHODS: PCR and enzymatic digestion were conducted to identify oral shedding of herpesviruses and its correlation with OM development in 31 patients. The samples were collected at three sites in the oral cavity and at 5 times during follow-up; two additional collections were made from patients who developed ulcerative OM. RESULTS: HSV-1, EBV, CMV, HHV-6A, HHV-6B, and HHV-7 were detected in 4.97%, 16.02%, 4.41%, 2.20%, 3.31%, and 68% of the oral mucosal samples, respectively; 4.41%, 16.57%, 5.52%, 2.20%, 5.52%, and 63.53% of supragingival samples, respectively, and 4.41%, 18.23%, 2.76%, 1.65%, 2.75%, and 35.91% of subgingival samples, respectively. OM was diagnosed in 13 patients. The presence of HHV-7 in C1 (oral mucosa: p = 0.032) and C2 (supragingival: p = 0.009; subgingival: p = 0.002) was significantly increased in patients who developed OM, and patients exhibiting HHV-7 shedding in the oral cavity were 3.32-fold more likely to develop OM. CONCLUSIONS: Patients who developed OM showed higher HHV-7 shedding in the oral cavity at nadir (immediately prior to OM development), suggesting modifications to the inflammatory microenvironment. CLINICAL RELEVANCE: HHV-7 may be involved in oral dysbiosis in HSCT-related OM; enhanced understanding of its role in the pathogenesis of OM may lead to the development of strategies for managing and preventing this common side effect of alloHSCT.

Intratumoral Genetic Heterogeneity in Rectal Cancer: Are Single Biopsies representative of the entirety of the tumor?

OBJECTIVE: Demonstrate intratumoral genetic heterogeneity in rectal cancer. BACKGROUND: Several clinical management decisions in rectal cancer may be influenced by pretreatment biopsy information. However, in the setting of significant intratumoral heterogeneity, biopsies may not be representative of the entirety of the tumor and limit the reliability of the information provided from them for clinical decision management. METHODS: Three fragments from a single rectal adenocarcinoma were chosen for whole-exome sequencing followed by mutation detection analysis. About 25 Gb of unambiguously mapped sequences were generated for each sample resulting in a median fold-coverage of 35x. Captured sequences mapped to the reference human genome were then used for the detection of somatic point mutations. RESULTS: Overall, 193 unique somatic point mutations were identified. Only 53 (27%) of these were shared by all three fragments, including known genes involved in early phases of the adenoma-carcinoma sequence (such as, APC). Approximately, 115 (59%) mutations were exclusively present in only one of the fragments, including mutations in "driver" genes (DNAH12). Jaccard distances showed a median distance of 0.603 for pair-wise comparison of fragments indicating significant heterogeneity between them. CONCLUSIONS: Considerable intratumoral heterogeneity is present among naive rectal cancers. The majority of point mutations detected in different fragments from rectal cancers are frequently unique to a single fragment. These findings support that gene mutations found on single pretreatment biopsies will not necessarily be representative of mutations present in the entirety of the tumor and therefore may limit the utility of the biological information provided by single biopsy fragments for clinical management decisions.

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin-fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G>T:A and C:G>A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct.

Light/Dark Environmental Cycle Imposes a Daily Profile in the Expression of microRNAs in Rat CD133(+) Cells.

The phenotype of primary cells in culture varies according to the donor environmental condition. We recently showed that the time of the day imposes a molecular program linked to the inflammatory response that is heritable in culture. Here we investigated whether microRNAs (miRNAs) would show differential expression according to the time when cells were obtained, namely daytime or nighttime. Cells obtained from explants of cremaster muscle and cultivated until confluence (∼20 days) presented high CD133 expression. Global miRNA expression analysis was performed through deep sequencing in order to compare both cultured cells. A total of 504 mature miRNAs were identified, with a specific miRNA signature being associated to the light versus dark phase of a circadian cycle. miR-1249 and miR-129-2-3p were highly expressed in daytime cells, while miR-182, miR-96-5p, miR-146a-3p, miR-146a-5p, and miR-223-3p were highly expressed in nighttime cells. Nighttime cells are regulated for programs involved in cell processes and development, as well as in the inflammation, cell differentiation and maturation; while daytime cells express miRNAs that control stemness and cytoskeleton remodeling. In summary, the time of the day imposes a differential profile regarding to miRNA signature on CD133(+) cells in culture. Understanding this daily profile in the phenotype of cultured cells is highly relevant for clinical outputs, including cellular therapy approaches. J. Cell. Physiol. 231: 1953-1963, 2016. © 2016 Wiley Periodicals, Inc.

Gene copy-number polymorphism caused by retrotransposition in humans.

The era of whole-genome sequencing has revealed that gene copy-number changes caused by duplication and deletion events have important evolutionary, functional, and phenotypic consequences. Recent studies have therefore focused on revealing the extent of variation in copy-number within natural populations of humans and other species. These studies have found a large number of copy-number variants (CNVs) in humans, many of which have been shown to have clinical or evolutionary importance. For the most part, these studies have failed to detect an important class of gene copy-number polymorphism: gene duplications caused by retrotransposition, which result in a new intron-less copy of the parental gene being inserted into a random location in the genome. Here we describe a computational approach leveraging next-generation sequence data to detect gene copy-number variants caused by retrotransposition (retroCNVs), and we report the first genome-wide analysis of these variants in humans. We find that retroCNVs account for a substantial fraction of gene copy-number differences between any two individuals. Moreover, we show that these variants may often result in expressed chimeric transcripts, underscoring their potential for the evolution of novel gene functions. By locating the insertion sites of these duplicates, we are able to show that retroCNVs have had an important role in recent human adaptation, and we also uncover evidence that positive selection may currently be driving multiple retroCNVs toward fixation. Together these findings imply that retroCNVs are an especially important class of polymorphism, and that future studies of copy-number variation should search for these variants in order to illuminate their potential evolutionary and functional relevance.

ICRmax: an optimized approach to detect tumor-specific interchromosomal rearrangements for clinical application.

Somatically acquired chromosomal rearrangements occur at early stages during tumorigenesis and can be used to indirectly detect tumor cells, serving as highly sensitive and tumor-specific biomarkers. Advances in high-throughput sequencing have allowed the genome-wide identification of patient-specific chromosomal rearrangements to be used as personalized biomarkers to efficiently assess response to treatment, detect residual disease and monitor disease recurrence. However, sequencing and data processing costs still represent major obstacles for the widespread application of personalized biomarkers in oncology. We developed a computational pipeline (ICRmax) for the cost-effective identification of a minimal set of tumor-specific interchromosomal rearrangements (ICRs). We examined ICRmax performance on sequencing data from rectal tumors and simulated data achieving an average accuracy of 68% for ICR identification. ICRmax identifies ICRs from low-coverage sequenced tumors, eliminates the need to sequence a matched normal tissue and significantly reduces the costs that limit the utilization of personalized biomarkers in the clinical setting.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling.

BACKGROUND: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines. METHODS: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform. RESULTS: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands. CONCLUSIONS: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.

The bladder microbiota is not significantly altered by intravesical BCG therapy.

INTRODUCTION: Bladder microbiota dysbiosis has been associated with several urological disorders. However, dysbiosis markers in bladder cancer have not been identified and little is known about the effect of Bacillus Calmette-Guérin (BCG) intravesical therapy on the bladder microbiota. In this study, we compared the bladder microbiota of patients with non-muscle-invasive bladder cancer (NMIBC) undergoing BCG therapy to nononcological controls. We also longitudinally analyzed the impact of BCG therapy on the bladder microbiota of NMIBC patients and addressed whether bladder microbiota is associated with BCG efficacy. METHODS: We collected catheterized urine samples from males with intermediate/high-risk NMIBC (cancer group, n = 32) or benign prostatic hyperplasia (control group, n = 41). The cancer group also provided urine samples during and after BCG induction. We used 16S rRNA gene sequencing to characterize the bladder microbiota. Bladder microbiota parameters, such as diversity and taxonomic composition, were compared between groups and associated with clinicopathological data and BCG efficacy. RESULTS: We observed no significant differences between the bladder microbiota of NMIBC patients and controls. BCG intravesical instillations did not significantly alter the bladder microbiota of NMIBC patients, and BCG was rarely detected in the bladder during and after BCG therapy. Microbiota diversity and overall composition before BCG induction did not influence disease persistence at 3 months. However, higher abundance of Lactobacillus, Streptococcus, and Cutibacterium in the pre-BCG bladder microbiota was associated with BCG effectiveness. CONCLUSION: We were unable to identify markers of bladder microbiota dysbiosis among male NMIBC patients. Moreover, we demonstrated for the first time using longitudinally collected samples that BCG cannot persist in the bladder microbiota nor significantly alter its diversity and composition. The associations found between bladder microbes and BCG efficacy highlight the potential of microbial-based therapeutic and risk-stratification strategies in the intermediate/high-risk NMIBC setting.

Moderate-intensity aerobic exercise training improves CD8+ tumor-infiltrating lymphocytes effector function by reducing mitochondrial loss.

Aerobic exercise training (AET) has emerged as a strategy to reduce cancer mortality, however, the mechanisms explaining AET on tumor development remain unclear. Tumors escape immune detection by generating immunosuppressive microenvironments and impaired T cell function, which is associated with T cell mitochondrial loss. AET improves mitochondrial content and function, thus we tested whether AET would modulate mitochondrial metabolism in tumor-infiltrating lymphocytes (TIL). Balb/c mice were subjected to a treadmill AET protocol prior to CT26 colon carcinoma cells injection and until tumor harvest. Tissue hypoxia, TIL infiltration and effector function, and mitochondrial content, morphology and function were evaluated. AET reduced tumor growth, improved survival, and decreased tumor hypoxia. An increased CD8+ TIL infiltration, IFN-γ and ATP production promoted by AET was correlated with reduced mitochondrial loss in these cells. Collectively, AET decreases tumor growth partially by increasing CD8+ TIL effector function through an improvement in their mitochondrial content and function.