Partial freezing of rat livers extends preservation time by 5-fold.

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

Blood-based biomarkers for the diagnosis and monitoring of gliomas.

Monitoring patient response to treatment is challenging for most cancers, but it is particularly difficult in glioblastoma multiform, the most common and aggressive form of malignant brain tumor. These tumors exhibit a high degree of heterogeneity which may not be reflected in a biopsy. To determine if the current standard of care is effective, glioma patients are monitored using MRI or CT scans, an effective but sometimes misleading approach due to the phenomenon of pseudoprogression. As such, there is incredible need for a minimally invasive "liquid biopsy" to assist in molecularly characterizing the tumors while also aiding in the identification of true progression in glioblastoma. This review details the status and potential impact for circulating tumor cells, extracellular vesicles, ctDNA, and ctRNA, putative circulating biomarkers found in the blood in glioblastoma patients. As mutation-based therapy becomes more prevalent in gliomas, blood-based analyses may offer a non-invasive method of identifying mutations. The ability to obtain serial "liquid biopsies" will provide unique opportunities to study the evolution of tumors and mechanisms of treatment resistance and monitor for mutational changes in response to therapy.

Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common tumor of the oral cavity and has been associated with poor prognosis. Scarce prognostic markers are available for guiding treatment and/or sub-classifying patients. This study aims to identify biomarkers by searching for genes whose expression is increased or decreased during tumor progression (through T1 to T4 stages). Thirty-six samples from all tumor size stages (from T1 to T4) were analyzed using cDNA microarrays. Selected targets were analyzed by immunohistochemistry and in circulating tumor cells by immunofluorescence and Nanostring. Correlation was shown between PD-L1 and tumor size and lymph node metastasis, HOXB9 and tumor size, BLNK and perineural invasion, and between ZNF813 and perineural invasion. PD-L1 positivity was an independent prognostic factor in this cohort (p = 0.044, HH = 0.426). In CTCs from patients with locally advanced OSCC, we found a strong cytoplasmatic expression of PD-L1. PD-L1 is a ligand of PD-1 and is believed to limit T cell activity in inflammatory responses and limit autoimmune diseases. We demonstrated an important role for PD-L1 in primary tumors according to tumor size, and in disease specific survival. Therefore, we could further determine individuals with PD-L1+ CTCs, and possibly follow treatment using CTCs.

CD44+/CD133+ immunophenotype and matrix metalloproteinase-9: Influence on prognosis in early-stage oral squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the expression of CD44 and/or CD133 immunophenotypes and the associated effects of matrix metalloproteinase-9 (MMP-9) in early-stage oral squamous cell carcinomas (SCC) to assess their influence on tumor prognosis. METHODS: The following data were derived from 150 patients: age, sex, primary anatomic site, smoking status, alcohol intake, recurrence, metastases, histological classification, treatment, disease-free survival (DFS), and overall survival (OS). Immunohistochemical study of CD44, CD133, and MMP-9 expression was performed on a tissue microarray of 150 paraffin blocks of oral SCCs. RESULTS: The predominant immunophenotype identified to exhibit a significant correlation with MMP-9 was the CD44+/CD133+. Multivariate analyses identified a significant correlation of OS with surgical treatment and with CD44+/CD133+ immunophenotype. CONCLUSION: This investigation demonstrated the prognostic importance of CD44/CD133 expression, which can help improve the prognostic value of surgical treatment for oral SCCs when diagnosed in early stages.

BRCA1 and γH2AX as independent prognostic markers in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCC) are believed to originate from sequential mutations that can develop as a consequence of genetic instability acquired over time. BRCA1 are linked to DNA recombination and repair processes, being of importance for its role in regulation of RAD51 and H2AX (γH2AX). The aim of this study was to investigate the relationship between BRCA1 expression status and evaluate its prognostic impact. We selected from 150 OSCC patients, and evaluated BRCA1 expression in OSCC by immunohistochemistry and qRT-PCR, comparing its expression with homologous recombination markers (RAD51, γH2AX and p53), clinicopathological and survival data. Expression of BRCA1 was observed in 61 cases (43.88%) and was related to tumor size (T stage) (p=0.001), and gender (p=0.017). mRNA from BRCA1 showed a borderline relationship with perineural invasion (p=0.053). BRCA1 [p=0.030; HR: 2.334 (C.I.: 1.087-5.012)], γH2AX [p=0.045; HR: 0.467 (C.I.: 0.222-0.628)] and gender [p=0.001; HR: 10.386 [(C.I.: 2.679-10.623)] were independent prognostic factors for DSS. BRCA1 and γH2AX expression by OSCC cells are associated with reduced overall survival time, independent of other variables in patients, as well as gender, and our findings shed some light about DSB markers in OSCC and its role as prognostic factors.

The relationship between lymphatic vascular density and vascular endothelial growth factor A (VEGF-A) expression with clinical-pathological features and survival in pancreatic adenocarcinomas.

BACKGROUND: Pancreatic cancer is a rare tumor with an extremely low survival rate. Its known risk factors include the chronic use of tobacco and excessive alcohol consumption and the presence of chronic inflammatory diseases, such as pancreatitis and type 2 diabetes. Angiogenesis and lymphangiogenesis, which have been the focus of recent research, are considered prognostic factors for cancer development. Knowing the angiogenic and lymphangiogenic profiles of a tumor may provide new insights for designing treatments according to the different properties of the tumor. The aim of this study was to evaluate the density of blood and lymphatic vessels, and the expression of VEGF-A, in pancreatic adenocarcinomas, as well as the relationship between blood and lymphatic vascular density and the prognostically important clinical-pathological features of pancreatic tumors. METHODS: Paraffin blocks containing tumor samples from 100 patients who were diagnosed with pancreatic cancer between 1990 and 2010 were used to construct a tissue microarray. VEGF expression was assessed in these samples by immunohistochemistry. To assess the lymphatic and vascular properties of the tumors, 63 cases that contained sufficient material were sectioned routinely. The sections were then stained with the D2-40 antibody to identify the lymphatic vessels and with a CD34 antibody to identify the blood vessels. The vessels were counted individually with the Leica Application Suite v4 program. All statistical analyses were performed using SPSS 18.0 (Chicago, IL, USA) software, and p values ≤ 0.05 were considered significant. RESULTS: In the Cox regression analysis, advanced age (p=0.03) and a history of type 2 diabetes (p=0.014) or chronic pancreatitis (p=0.02) were shown to be prognostic factors for pancreatic cancer. Blood vessel density (BVD) had no relationship with clinical-pathological features or death. Lymphatic vessel density (LVD) was inversely correlated with death (p=0.002), and by Kaplan-Meyer survival analysis, we found a significant association between low LVD (p=0.021), VEGF expression (p=0.023) and low patient survival. CONCLUSIONS: Pancreatic carcinogenesis is related to a history of chronic inflammatory processes, such as type 2 diabetes and chronic pancreatitis. In pancreatic cancer development, lymphangiogenesis can be considered an early event that enables the dissemination of metastases. VEGF expression and low LVD can be considered as poor prognostic factors as tumors with this profile are fast growing and highly aggressive. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5113892881028514.

Relationship between B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase IIIß (topoIIIß) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIIIß, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA-1 (p= 0.003), H2AX (p=0.024) and topoIIIß (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Topoisomerase expression in oral squamous cell carcinoma: relationship with cancer stem cells profiles and lymph node metastasis.

BACKGROUND: The relationship between predictive proteins and tumors presenting cancer stem cells (CSCs) profiles in oral tumors is still poorly understood. This study aims to identify the relationship between topoisomerases I, IIα, and IIIα and putative CSCs immunophenotype in oral squamous cell carcinoma (OSCC) and determine its influence on prognosis. METHODS: The following data were retrieved from 127 patients: age, gender, primary anatomic site, smoking and alcohol intake, recurrence, metastases, histologic classification, treatment, and survival. An immunohistochemical study for topoisomerases I, IIα, and IIIα was performed in a tissue microarray containing 127 paraffin blocks of OSCCs. RESULTS: In univariate analysis, topoisomerases expression showed significant differences according to CSCs profiles and p53 immunoexpression, but not with survival. Topoisomerases IIα and IIIα also showed significant relationship with lymph node metastasis. The multivariate test confirmed these associations. CONCLUSIONS: The results that all topoisomerases correlates with OSCC CSCs may indicate a role for topoisomerases in head and neck carcinogenesis. Notwithstanding, it is plausible that other members of topoisomerases family could represent novel therapeutical targets in oral squamous cell carcinoma.

Carcinoma mucoepidermoide de las glándulas salivares en Brasil: resultados clinicopatológicos y una breve revisión / Mucoepidermoid carcinoma of the salivary glands in Brazil: clinicopathological outcomes and a brief review

The biological features and the clinical behavior of the mucoepidermoid carcinoma are varied and not known yet. The aim of present paper was to analyze the potential prognostic factors affecting the survival of patients diagnosed with primary mucoepidermoid carcinoma of head and neck. A retrospective study was conducted in 16 patients treated between 1990 and 2008 in the General Hospital of Riberirao Preto, USP Medicine School, Brazil. The following variables were studied: age, sex, anatomical location, tumor size, clinical stage, histological degree, relapse, metastasis, involved surgical edges ant treatment on the clinical-pathological results. The survival curves were designed using the Kaplan-Meier method and the statistic analysis was made using the log-rank test. The 68.7 percent of patients was of male sex, all patients were between 13 and 83 years old. The 75 percent of tumors was located in the great salivary glands, the 56.3 percent in the parotid glands ones, the mucoepidermoid carcinomas of low degree and of II stage were the 37.5 percent. The surgical resection was carried out in all patients. The follow-up period in present study fluctuates between 6 and 217 months. The general rate of 5- y years or 10-years survival was of 85.6 percent whereas the rates of disease-free survival were of 81.8 percent at 5 years and of 68.2 percent at 10 years. The were statistically significant influences of the tumor size (p = 0.05), presence of metastasis (p = 0.04) and of the primary anatomical location (p = 0.04) on the rates of disease-free survival. The results obtained show the significance of the primary anatomical location of the tumor, of its size and of the presence of metastasis in the survival of mucoepidermoid carcinomas(AU)

Las características biológicas y el comportamiento clínico del carcinoma mucoepidermoide son muy variados y aún poco conocidos. El propósito de este estudio fue analizar los factores pronósticos que puedan afectar la supervivencia de los pacientes con diagnóstico de carcinoma mucoepidermoide primario de cabeza y cuello. Se realizó un estudio retrospectivo de 16 pacientes tratados entre 1990 y 2008 en el Hospital General de Ribeirao Preto, Escuela de Medicina USP, Brasil. Se estudiaron las variables: edad, sexo, localización anatómica, tamaño del tumor, estadio clínico, grado histológico, recidiva, metástasis, bordes quirúrgicos comprometidos y tratamiento, sobre los resultados clínico-patológicos. Las curvas de supervivencia fueron construidas utilizando el método de Kaplan-Meier y el análisis estadístico fue realizado mediante la prueba del log-rank. Se constató 68,7 por ciento de pacientes del sexo masculino, todos los pacientes comprendidos en las edades entre 13 y 83 años. El 75 por ciento de los tumores se localizó en las glándulas salivales mayores, 56,3 por ciento en parótida, los carcinomas mucoepidermoides de bajo grado y estadio II con 37,5 por ciento. La resección quirúrgica fue realizada en todos los pacientes. El período de seguimiento en este estudio varió entre 6 y 217 meses. La tasa general de supervivencia, tanto a los 5 como a los 10 años fue de 85,6 por ciento, mientras que las tasas de supervivencia libre de enfermedad fueron de 81,8 por ciento a los 5 años y de 68,2 por ciento a los 10 años. Se demostró la existencia de influencias estadísticamente significativas del tamaño del tumor (p = 0,05), presencia de metástasis (p = 0,04), y de la localización anatómica primaria (p = 0,04) sobre las tasas de supervivencia libre de enfermedad. Los resultados obtenidos demuestran la importancia de la localización anatómica primaria del tumor, de su tamaño y de la presencia de metástasis, en la supervivencia de los carcinomas mucoepidermoides(AU)

Carcinoma mucoepidermoide de glándulas salivales en Brasil: relación clinicopatológica / Mucoepidermoid carcinoma of the salivary glands in Brazil: clinicopathological outcomes

The biological features and clinical behavior of mucoepidermoid carcinomas are widely variable and poorly understood. This study aimed to investigate prognostic factors that may affect survival in patients with a primary diagnosis of head and neck mucoepidermoid carcinomas. The effects of age, gender, anatomic localization, tumor size, clinical stage, histological grade, recurrence, metastasis, compromised surgical margins and treatment on clinicopathological outcomes were investigated. Survival curves were generated using the Kaplan-Meier method and analyses were performed using the log rank test. A total of 16 cases were analyzed over a period of 18 years; males were 68.7 percent, with ages ranging from 13 to 83 years. The 75 percent of the tumors developed in the major salivary glands, 56.3 percent in the parotid gland and they were predominantly classified as stage II 37.5 percent and low-grade lesions 37.5 percent at diagnosis. Surgical resection was performed in all patients. The follow-up period in this study ranged from 6 to 217 months. The 5 and 10-year overall survival rates were both 85.6 percent. Disease-free survival rates were 81.8 percent (5 years) and 68.2 percent (10 years). There were statistically significant effects of tumor size (p= 0.05), metastasis (p= 0.04) and primary anatomic localization (p= 0.04) on disease-free survival rates. Through a long follow-up period in present study we could highlight the relevance of primary anatomical site, tumor size and metastasis as useful prognostic factors that may affect survival in patients with a primary diagnosis of head and neck mucoepidermoid carcinomas(AU)

Las características biológicas y el comportamiento clínico del carcinoma mucoepidermoide son muy variados y aún poco conocidos. El propósito de este estudio fue investigar los factores pronósticos que puedan afectar la supervivencia de los pacientes con diagnóstico primario de carcinoma mucoepidermoide de cabeza y cuello. Se estudiaron la edad, el sexo, la localización anatómica, el tamaño del tumor, el estadio clínico, el grado histológico, la recidiva, la metástasis, los bordes quirúrgicos comprometidos y el tratamiento, sobre los resultados clínico-patológicos. Las curvas de supervivencia fueron construidas con el método de Kaplan-Meier y el análisis estadístico fue realizado mediante la prueba del log-rank. Fueron analizados 16 casos durante un periodo de 18 años. Se constató un 68,7 por ciento de pacientes del sexo masculino y de edades comprendidas entre los 13 y los 83 años. El 75 por ciento de los tumores se localizó en las glándulas salivales mayores, el 56,3 por ciento en parótida y las clasificaciones predominantes en el momento del diagnóstico fueron lesiones de bajo grado y estadio II con un 37,5 por ciento. La resección quirúrgica fue realizada en todos los pacientes. El periodo de seguimiento en este estudio varió entre 6 y 217 meses. La tasa general de supervivencia, tanto a los 5 como a los 10 años fue de 85,6 por ciento, mientras que las tasas de supervivencia libre de enfermedad fueron de 81,8 por ciento a los 5 años y de 68,2 por ciento a los 10 años. Se demostró la influencia estadísticamente significativa del tamaño del tumor (p= 0,05), la presencia de metástasis (p= 0,04) y de la localización anatómica primaria (p= 0,04) sobre las tasas de supervivencia libre de enfermedad. Los resultados obtenidos demostraron la importancia de la localización anatómica primaria del tumor, de su tamaño y de la presencia de metástasis, en la supervivencia de los pacientes con diagnóstico primario de carcinoma mucoepidermoide(AU)

The role of tumor hypoxia in MUC1-positive breast carcinomas.

Mucin 1 (MUC1) is a glycoprotein that is expressed on apical cell membranes in a variety of normal tissues. MUC1 is involved in cell signaling, inhibition of cell-cell and cell matrix adhesion, apoptosis, proliferation, and transcription. Hypoxia is an important factor that promotes cancer metastasis and stimulates angiogenesis and tumor progression. Hypoxia inducible factor 1 (HIF-1α) and carbonic anhydrase IX (CAIX) are two molecules that are involved in this process. The role of hypoxia in MUC1+ invasive ductal breast carcinomas is not well established. In this study, the expression of MUC1 was correlated with the hypoxia-associated markers HIF-1α and CAIX, as well as several immunohistochemical markers and clinicopathologic features of prognostic significance in 243 invasive ductal carcinomas. MUC1 was overexpressed in 37.0% of patients and correlated with the expression of estrogen receptor (p = 0.0001), progesterone receptor (p = 0.0001), HIF-1α (p = 0.006), VEGF (p = 0.024), and p53 (p = 0.025). In breast cancer, MUC1 expression has been associated with increased degradation of inhibitor of NF-κB (IκBα), driving NF-κB to the nucleus and blocking apoptosis and promoting cell survival. We analyzed NF-κB expression in MUC1+ breast carcinoma and found a very significant relationship between these proteins (p = 0.0001). Our findings indicate that MUC1 may play a role in the regulation of hormone receptors by increasing the inactivation of p53 and targeting NF-κB to the nucleus. Our data also support the notion that activation of HIF-1α in MUC1+ breast carcinomas may modulate VEGF expression, allowing a metabolic adaptation to hypoxia.

Differential expression of HIF-1α in CD44+CD24-/low breast ductal carcinomas.

BACKGROUND: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1α expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44+CD24-/low immunophenotype. METHODS: Immunohistochemical analysis of CD44+CD24-/low expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). RESULTS: In univariate analysis, CD44+CD24-/low expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44+CD24-/low immunophenotype showed relationship with HIF-1α (p = 0.039) and negativity for HER-2 (p = 0.013). CONCLUSION: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44+CD24-/low positive breast tumors.

Prognostic factors in patients with malignant salivary gland neoplasms in a Brazilian population.

Due to the difficulty of follow-up for long periods, information about the survival rates of malignant salivary gland tumors is deficient in the global scientific literature. This study was aimed at investigating the epidemiological profile and prognostic factors that might affect survival in patients with primary malignant salivary gland tumors in Brazil. Patients were investigated regarding histopathological subtypes, age, gender, anatomic localization, smoking and alcohol intake, tumor size, clinical stage, histological grade, recurrence, metastasis, and treatment on clinicopathological outcomes. Survival curves were generated using the Kaplan-Meier method, and both univariate and multivariate analyses were performed using the log rank test and Cox regression, respectively. A total of 63 cases were analyzed, females being slightly predominant (50.8%), with ages ranging from 13 to 87 years. The most common diagnosis was adenoid cystic carcinoma and the most affected anatomical location was the parotid. Tumors were predominantly classified as stage I and high-grade at the diagnosis. The 5- and 10-year overall survival rates were 84.6% and 74.7%, respectively. Disease-free survival (DFS) rates were 71.6% (5 years) and 56.6% (10 years). Univariate analysis showed significant effects of tumor size and clinical stage on the DFS (P<0.0001 for both), and Cox regression analysis confirmed clinical stage as an independent prognostic factor (P = 0.035). Our results highlight the relevance of clinical stage as an independent prognostic parameter for malignant salivary gland tumors.

Significance of topoisomerase IIIß expression in breast ductal carcinomas: strong associations with disease-specific survival and metastasis.

Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase IIIß, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase IIIß in breast cancer and its relationships with clinicopathologic features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase IIIß, estrogen receptor, progesterone receptor, HER-2, BRCA-1, p53, and Ki67. Immunostaining for topoisomerase IIIß was found in 33.9% of breast carcinomas, and immunopositivity was correlated with distant metastasis (P = .036) and death (P = .006). Decreased expression of topoisomerase IIIß correlated with low expression of Ki67 (P < .001) and negativity for HER-2 (P < .001), BRCA-1 (P = .001), and p53 (P < .001). In the multivariate analysis, topoisomerase IIIß expression was a significant predictor of survival (hazard ratio, 3.006 [95% confidence interval, 1.582-5.715]; P = .001). In conclusion, topoisomerase IIIß expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival.