Manal Gabril

Exploring the role of miRNAs in renal cell carcinoma progression and metastasis through bioinformatic and experimental analyses

Metastasis results in most of the cancer deaths in clear cell renal cell carcinoma (ccRCC). MicroRNAs (miRNAs) regulate many important cell functions and play important roles in tumor development, metastasis and progression. In our previous study, we identified a miRNA signature for metastatic RCC. In this study, we validated the top differentially expressed miRNAs on matched primary and metastatic ccRCC pairs by quantitative polymerase chain reaction. We performed bioinformatics analyses including target prediction and combinatorial analysis of previously reported miRNAs involved in tumour progression and metastasis. We also examined the co-expression of the miRNAs clusters and compared expression of intronic miRNAs and their host genes. We observed significant dysregulation between primary and metastatic tumours from the same patient. This indicates that, at least in part, the metastatic signature develops gradually during tumour progression. We identified metastasis-dysregulated miRNAs that can target a number of genes previously found to be involved in metastasis of kidney cancer as well as other malignancies. In addition, we found a negative correlation of expression of miR-126 and its target vascular endothelial growth factor (VEGF)-A. Cluster analysis showed that members of the same miRNA cluster follow the same expression pattern, suggesting the presence of a locus control regulation. We also observed a positive correlation of expression between intronic miRNAs and their host genes, thus revealing another potential control mechanism for miRNAs. Many of the significantly dysregulated miRNAs in metastatic ccRCC are highly conserved among species. Our analysis suggests that miRNAs are involved in ccRCC metastasis and may represent potential biomarkers.

Informatics for practicing anatomical pathologists: marking a new era in pathology practice

Informatics can be defined as using highly advanced technologies to improve patient diagnosis or management. Pathology informatics had evolved as a response to the overwhelming amount of information that was available, in an attempt to better use and maintain them. The most commonly used tools of informatics can be classified into digital imaging, telepathology, as well as Internet and electronic data mining. Digital imaging is the storage of anatomical pathology information, either gross pictures or microscopic slides, in an electronic format. These images can be used for education, archival, diagnosis, and consultation. Virtual microscopy is the more advanced form of digital imaging with enhanced efficiency and accessibility. Telepathology is now increasingly becoming a useful tool in anatomical pathology practice. Different types of telepathology communications are available for both diagnostic and consultation services. The spectrum of applications of informatics in the field of anatomical pathology is broad and encompasses medical education, clinical services, and pathology research. Informatics is now settling on solid ground as an important tool for pathology teaching, with digital teaching becoming the standard tool in many institutions. After a slow start, we now witness the transition of informatics from the research bench to bedside. As we are moving into a new era of extensive pathology informatics utilization, several challenges have to be addressed, including the cost of the new technology, legal issues, and resistance of pathologists. It is clear from the current evidence that pathology informatics will continue to grow and have a major role in the future of our specialty. However, it is also clear that it is not going to fully replace the human factor or the regular microscope.

Microvascular density as an independent predictor of clinical outcome in renal cell carcinoma: an automated image analysis study

Tumor microvascular density (MVD) has been shown to correlate with the aggressiveness of several cancers. With the introduction of targeted anti-angiogenic therapy, assessment of MVD has the potential not only as a prognostic but also as a therapeutic marker. The significance of tumor vascularity in clear cell renal cell carcinoma (ccRCC) has been debated, with studies showing contradictory results. Previous studies were limited by manual quantification of MVD within a small area of tumor. Since then, the validity of this method has been questioned. To avoid the inaccuracies of manual quantification, we employed a computerized image analysis, which allowed assessment of large areas of tumor and adjacent normal tissue. The latter was used as an internal reference for normalization. MVD and vascular endothelial growth factor (VEGF) were assessed in 57 cases of ccRCC. Sections were immunostained for CD34 and VEGF. Areas of ccRCC and normal kidney medulla were analyzed within scanned images using software that counted CD34-positive vessels and measured the intensity of VEGF staining. We obtained unadjusted values from tumoral areas and calculated adjusted values as tumor/normal ratios. Unadjusted MVD had no association with clinical outcome. However, similarly to tumor stage, higher adjusted MVD was associated with shorter disease-free survival (log-rank P=0.037, Cox P=0.02). This was significant in univariate and multivariate analyses. MVD did not correlate with tumor stage, pointing to its independent prognostic value. As expected due to the known molecular abnormalities in ccRCC, most tumors showed higher VEGF expression than normal tissue. Higher adjusted VEGF was associated with high tumor grade (P=0.049). The finding of increased MVD as an independent marker of tumor aggressiveness may prove useful in the development of new tests for prognostic and therapeutic guidance. Digital techniques can provide more accurate assessment of immunomarkers and may reveal less obvious associations.

Evaluation and prognostic significance of human tissue kallikrein-related peptidase 6 (KLK6) in colorectal cancer

The prognosis of patients with colorectal cancer (CRC) is assessed through conventional clinicopathological parameters, which are not always accurate. Members of the human kallikrein-related peptidases gene family represent potential cancer biomarkers. The aim of this study was to investigate the expression of human tissue kallikrein-related peptidase 6 (KLK6) by immunohistochemistry in CRC to correlate this expression with various histopathological and clinical variables, and to evaluate its significance as a predictor of disease outcome. KLK6 expression was evaluated by immunohistochemistry and an expression score was calculated for each case. In CRC, KLK6 expression was decreased compared to normal colonic mucosa. A statistically significant, positive association was observed between KLK6 and tumor stage (p=0.036), lymph node metastases (p=0.030), and liver metastases (p=0.025). Univariate analysis showed that KLK6 expression and stage had statistically significant correlation with disease-free survival (p=0.045 and p<0.001, respectively) and overall survival (p=0.027 and p<0.001, respectively). Cox multivariate analysis showed that KLK6 expression was an independent predictor of unfavorable overall survival (p=0.041). Kaplan-Meier survival curves showed that KLK6-positive patients have statistically significant lower disease-free and overall survival. In conclusion, KLK6 immunostaining is an independent prognostic marker in patients with CRC.

Assessment of the prognostic significance of endoglin (CD105) in clear cell renal cell carcinoma using automated image analysis

The behavior of clear cell renal cell carcinoma can be difficult to predict. Angiogenesis has proven to be a useful prognostic indicator in different malignancies. Endoglin (CD105) is a new marker of angiogenesis found to have prognostic utility in various tumors. Here, we provide the first automated digital assessment of intratumoral microvascular density in clear cell renal cell carcinoma using endoglin and CD31 and assess their utility as predictors of clinical outcome. Both endoglin and CD31 expression showed association with advanced tumor stage (P = .025 and P = .011, respectively). There was a significant correlation between CD31 and tumor grade (P = .034). Kaplan-Meier survival curves showed that patients with higher endoglin expression had significantly shorter progression-free survival (P = .010). Patients with higher CD31 expression tended to have a worse prognosis, although this was not statistically significant (P = .082). In univariate analysis using endoglin as a continuous variable, increased endoglin was strongly associated with reduced survival (hazard ratio, 1.74; 95% CI, 1.39-2.18; P = <.001). CD31 also correlated with poor outcomes (hazard ratio, 1.52; 95% CI, 1.24-1.86; P = .001). There was no correlation between CD31 and endoglin expression (r = -0.090, P = .541). Receiver operating characteristic analysis showed the area under the curve to be 0.749 for endoglin and 0.550 for CD31. In conclusion, increased endoglin and CD31 expression are associated with a higher tumor stage and decreased progression-free survival. Our automated approach overcomes many limitations of manual quantification. Advances in digital assessment of immunohistochemical markers can be helpful in standardizing the evaluation of tumor biomarkers.

The clinical significance of in-depth pathological assessment of extraprostatic extension and margin status in radical prostatectomies for prostate cancer.

Despite recent Level 1 evidence on the benefits of adjuvant radiotherapy for locally advanced prostate cancer (PCa), the timing and decision to administer adjuvant radiotherapy post-radical prostatectomy (post-RP) remains debatable, particularly for patients with focal extraprostatic extension (EPE) and/or focal positive surgical margins (PSMs). In this study, we assess the utility of detailed pathological assessment of EPE and PSM, as this may influence the criteria for instituting adjuvant radiotherapy. A total of 148 RP cases (1993–2001) were identified retrospectively as having EPE and/or PSM. All slides were re-reviewed, incorporating recent proposals by the Collage of American Pathologists (CAP) for the reporting of EPE and PSM, and correlated with clinical data. Both EPE and PSM were found to be independent predictors of biochemical failure (BCF); however, only EPE was associated with metastasis and death. BCF was also more likely to be associated with cases that had non-focal EPE than focal EPE. Similarly, non-focal PSM cases had a significantly higher risk of BCF than focal cases. Our study confirms the value of detailed pathological assessment of EPE and PSM post-RP. The results support the concept of selective adjuvant radiotherapy in patients with EPE and PSM, based on focality and extent.

A rare case of recurrent urinary obstruction and acute renal failure from cystitis cystica et glandularis

We report a rare case of recurrent florid cystitis cystica et glandularis (CCEG), common type, causing obstruction of the left ureterovesicle junction (UVJ) leading to renal colic and hydronephrosis. A 43-year-old man was admitted with renal colic, left UVJ obstruction, left hydronephrosis and azotemia. Cystoscopy showed a >4-cm bladder lesion compressing the left UVJ. Transurethral resection of the bladder tumour (TURBT) was performed and pathology revealed the lesion as CCEG. Two months later, the CCEG recurred and caused left UVJ obstruction a second time, requiring TURBT.

An update on the molecular pathology of urinary bladder tumors

Urothelial carcinoma is the fourth most common tumors after prostate cancer, lung, and colorectal carcinoma but the second most common urologic malignancy. Urothelial carcinoma composed more than 90% of bladder tumors while squamous cell carcinoma and adenocarcinomas composed 5% and 2% respectively. The intense research involving the different molecular aspects of bladder cancer has provided a great insight into identifying more about molecular profiling and pathways of bladder cancer. In this review, we will highlight the general concepts of the molecular features; profiling and classification as well as the molecular pathways for bladder carcinomas, especially urothelial carcinoma. Also, we will discuss the advances of molecular biomarkers for screening, early diagnosis, surveillance and potential prognosis of urothelial carcinoma of the bladder. Studies showed that accumulation of genetic alterations involving the clonal expansion of altered cells with growth advantages through sequential multi-step pathways results in progression of bladder tumors. The accumulated research data from literature has revealed that the genomic signatures of urothelial carcinoma are required to subclassify bladder cancer into genetically distinct subgroups. These findings could improve the understating of pathogenesis as well as will provide new therapeutic modules e.g. targeted therapy.

INFLUENZA A H1N1, MICROSCOPIC POLYANGIITIS AND PULMONARY HAEMORRHAGE

The pathogenesis of microscopic polyangiitis (MPA) is not entirely understood, nor the numerous triggers that lead to MPA relapses. This case is a relapse of MPA likely secondary to an infectious aetiology. In November 2009, a 42-year-old man presented with a 48-hour history of fever, vomiting, diarrhoea and dyspnoea. He was diagnosed with MPA in July 2009, with pauciimmune segmental non-granulomatous necrotizing glomerulonephritis (peak creatinine 518 mmol/L), pulmonary haemorrhage and p-ANCA 288 U/mL (Fig. 1). He was treated with pulse steroids tapered to 5 mg/day and monthly intravenous cyclophosphamide. Examination revealed postural hypotension and a temperature of 39.4°C. Chest radiography showed opacities in the left lower lobe, which was similar to July At the time of his presentation our institution was experiencing an outbreak of H1N1. Differential diagnoses included multifactorial pneumonia, H1N1 influenza and MPA exacerbation. Cultures were taken, and levofloxacin, oseltamivir and methylprednisolone were initiated. Within 24 h, he became hypoxic and developed worsening cough with haemoptysis. Haemoglobin fell to 75 g/L and opacification of the left lower, right middle and lower lobes were noted. A course of seven plasma exchanges was started on day 2. The H1N1 swab was positive and p-ANCA titre was elevated. The patient was discharged day 9, on prednisone 60 mg and monthly cyclophosphamide. Birck et al. described four cases of new or relapsing ANCAassociated vasculitis following influenza vaccination. Stassen et al., however, concluded that influenza vaccination does not result in an increase in relapses in patients with ANCA-associated vasculitis. The conflicting literature suggests that the pathogenesis is poorly understood and the question of an influenza-associated immune trigger causing a relapse in ANCA-associated vasculitis still exists. In our case influenza A H1N1 infection was associated with a development of pulmonary haemorrhage; in a patient with known MPA. In order to ensure optimal outcomes, both diagnoses needed to be considered and treated. In patients with a history of vasculitis it is important to consider relapse when presenting with a new infection, including influenza A, as well as consider a new diagnosis of vasculitis in the rare number of H1N1 cases that develop pulmonary haemorrhage and rapidly progressive kidney disease.

Urothelial Tumors: Moving from Morphology to Biology

The fourth most common tumour of bladder is urothelial carcinoma. A great insight into the biology of the disease through molecular profiling and pathways analysis has been provided through the intense research involving the different molecular aspects of bladder cancer. Throughout this minireview, the general concepts of the molecular features of urothelial cancer will be reviewed. In addition, molecular-based classification for bladder carcinomas will be highlighted.