Molecular markers for urothelial bladder cancer prognosis: toward implementation in clinical practice

OBJECTIVES To summarize the current status of clinicopathological and molecular markers for the prediction of recurrence or progression or both in non-muscle-invasive and survival in muscle-invasive urothelial bladder cancer, to address the reproducibility of pathology and molecular markers, and to provide directions toward implementation of molecular markers in future clinical decision making. METHODS AND MATERIALS Immunohistochemistry, gene signatures, and FGFR3-based molecular grading were used as molecular examples focussing on prognostics and issues related to robustness of pathological and molecular assays. RESULTS The role of molecular markers to predict recurrence is limited, as clinical variables are currently more important. The prediction of progression and survival using molecular markers holds considerable promise. Despite a plethora of prognostic (clinical and molecular) marker studies, reproducibility of pathology and molecular assays has been understudied, and lack of reproducibility is probably the main reason that individual prediction of disease outcome is currently not reliable. CONCLUSIONS Molecular markers are promising to predict progression and survival, but not recurrence. However, none of these are used in the daily clinical routine because of reproducibility issues. Future studies should focus on reproducibility of marker assessment and consistency of study results by incorporating scoring systems to reduce heterogeneity of reporting. This may ultimately lead to incorporation of molecular markers in clinical practice.

Pathology: Classification and Immunoprofile

The classification of neuroendocrine neoplasms (NENs) has been evolving steadily over the last decades. Important prognostic factors of NENs are their proliferative activity and presence/absence of necrosis. These factors are reported in NENs of all body sites; however, the terminology as well as the exact rules of classification differ according to the location of the primary tumor. Only in gastroenteropancreatic (GEP) NENs a formal grading is performed. This grading is based on proliferation assessed by the mitotic count and/or Ki-67 proliferation index. In the lung, NEN grading is an intrinsic part of the tumor designation with typical carcinoids corresponding to neuroendocrine tumor (NET) G1 and atypical carcinoids to NET G2; however, the presence or absence of necrotic foci is as important as proliferation for the differentiation between typical and atypical carcinoids. Immunohistochemical markers can be used to demonstrate neuroendocrine differentiation. Synaptophysin and chromogranin A are, to date, the most reliable and most commonly used for this purpose. Beyond this, other markers can be helpful, for example in the situation of a NET metastasis of unknown primary, where a hormonal profile or a panel of transcription factors can give hints to the primary site. Many immunohistochemical markers have been shown to correlate with prognosis but are not used in clinical practice, for example cytokeratin 19 and KIT expression in pancreatic NETs. There is no predictive biomarker in use, with the exception of somatostatin receptor (SSTR) 2 expression for predicting the amenability of a tumor to in vivo SSTR targeting for imaging or therapy.

High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples.

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.

Prior knowledge of the clinical picture does not introduce bias in the histopathologic diagnosis of melanocytic skin lesions

A common debate among dermatopathologists is that prior knowledge of the clinical picture of melanocytic skin neoplasms may introduce a potential bias in the histopathologic examination. Histologic slides from 99 melanocytic skin neoplasms were circulated among 10 clinical dermatologists, all of them formally trained and board-certified dermatopathologists: 5 dermatopathologists had clinical images available after a 'blind' examination (Group 1); the other 5 had clinical images available before microscopic examination (Group 2). Data from the two groups were compared regarding 'consensus' (a diagnosis in agreement by ≥4 dermatopathologists/group), chance-corrected interobserver agreement (Fleiss' k) and level of diagnostic confidence (LDC: a 1-5 arbitrary scale indicating 'increasing reliability' of any given diagnosis). Compared with Group 1 dermatopathologists, Group 2 achieved a lower number of consensus (84 vs. 90) but a higher k value (0.74 vs. 0.69) and a greater mean LDC value (4.57 vs. 4.32). The same consensus was achieved by the two groups in 81/99 cases. Spitzoid neoplasms were most frequently controversial for both groups. The histopathologic interpretation of melanocytic neoplasms seems to be not biased by the knowledge of the clinical picture before histopathologic examination.

Assessment of English Language Learners Seeking a Graduate Degree in Speech Language Pathology

A shortage of bilingual/bicultural speech language pathologists may reflect a problem with recruitment and retention of bilingual/bicultural students. The purpose of the present study was to survey graduate training programs in speech language pathology to determine typical policies and practices concerning students who apply and are admitted as ELLs. With a growing number of ELL children needing services from a bilingual SLP, it seems that little is being done to address the issue. The problem may be with the reluctance of programs to not only accept ELL students, but there also seems to be a disinclination for any sort of training program to be established for these ELL students. Clinic directors were asked to complete a survey about ELLs seeking clinical training in speech language pathology. In particular, we were interested in obtaining information about whether clinical training programs a) provided opportunities for ELL to participate in clinic, b) assessed the English skills of these students, and c) provided remediation if these students English skills were judged to be less than proficient.

Association of pellicle growth morphological characteristics and clinical presentation of Mycobacterium tuberculosis isolates

Trehalose dimycolate (TDM) is a mycobacterial glycolipid that is released from the surface of virulent M. tuberculosis. We evaluated the rate of growth, colony characteristics and production of TDM by Mycobacterium tuberculosis strains isolated from different clinical sites. Since detergent removes TDM from organisms, we analyzed growth rate and colony morphology of 79 primary clinical isolates grown as pellicles on the surface of detergent free Middlebrook 7H9 media. The genotype of each had been previously characterized. TDM production was measured by thin layer chromatography on 32 of these isolates. We found that strains isolated from pulmonary sites produced large amounts of TDM, grew rapidly as thin spreading pellicles, showed early cording (<1 week) and climbed the sides of the dish. In contrast, the extrapulmonary isolates (lymph node and bone marrow) produced less TDM (p<0.01), grew as discrete patches with little tendency to spread or climb the walls (p<0.02). The Beijing pulmonary (BP) isolates produced more TDM than non Beijing pulmonary isolates. The largest differences were observed in Beijing strains. The Beijing pulmonary isolates produced more TDM and grew faster than the Beijing extrapulmonary isolates (p<0.01). This was true even when the pulmonary and extrapulmonary isolates were derived from the same clade. These growth characteristics were consistently observed only on the first passage after primary isolation. This suggests that the differences in growth rate and TDM production observed reflect differences in gene expression patterns of pulmonary and extrapulmonary infections, that Mycobacterium tuberculosis in the lung grows more rapidly and produces more TDM than it does in extrapulmonary sites. This provides new opportunities to investigate gene expression of Mycobacterium tuberculosis in human.^

TAZ AS A REGULATOR OF MESENCHYMAL TRANSFORMATION AND CLINICAL AGGRESSIVENESS IN GLIOMAS

Glioblastoma multiforme (GBM) is an aggressive, high grade brain tumor. Microarray studies have shown a subset of GBMs with a mesenchymal gene signature. This subset is associated with poor clinical outcome and resistance to treatment. To establish the molecular drivers of this mesenchymal transition, we correlated transcription factor expression to the mesenchymal signature and identified transcriptional co-activator with PDZ-binding motif (TAZ) to be highly associated with the mesenchymal shift. High TAZ expression correlated with worse clinical outcome and higher grade. These data led to the hypothesis that TAZ is critical to the mesenchymal transition and aggressive clinical behavior seen in GBM. We investigated the expression of TAZ, its binding partner TEAD, and the mesenchymal marker FN1 in human gliomas. Western analyses demonstrated increased expression of TAZ, TEAD4, and FN1 in GBM relative to lower grade gliomas. We also identified CpG islands in the TAZ promoter that are methylated in most lower grade gliomas, but not in GBMs. TAZ-methylated glioma stem cell (GSC) lines treated with a demethylation agent showed an increase in mRNA and protein TAZ expression; therefore, methylation may be another novel way TAZ is regulated since TAZ is epigenetically silenced in tumors with a better clinical outcome. To further characterize the role of TAZ in gliomagenesis, we stably silenced or over-expressed TAZ in GSCs. Silencing of TAZ decreased invasion, self-renewal, mesenchymal protein expression, and tumor-initiating capacity. Over-expression of TAZ led to an increase in invasion, mesenchymal protein expression, mesenchymal differentiation, and tumor-initiating ability. These actions are dependent on TAZ interacting with TEAD since all these effects were abrogated with TAZ could not bind to TEAD. We also show that TAZ and TEAD directly bind to mesenchymal gene promoters. Thus, TAZ-TEAD interaction is critically important in the mesenchymal shift and in the aggressive clinical behavior of GBM. We identified TAZ as a regulator of the mesenchymal transition in gliomas. TAZ could be used as a biomarker to both estimate prognosis and stratify patients into clinically relevant subgroups. Since mesenchymal transition is correlated to tumor aggressiveness, strategies to target and inhibit TAZ-TEAD and the downstream gene targets may be warranted in alternative treatment.

Reflections: Historical Perspectives on Pathology in Houston and Galveston

“This account of pathology in the Houston and Galveston area … examines important themes in the development of pathology in this area, using selected details from the careers of individuals and institutions to illustrate how pathologists, as practitioners, teachers, and researchers, dealt with the challenges they faced in finding and keeping a niche for pathology in the medical world.” - Preface This book was written to commemorate the 50th anniversary of Houston Society of Clinical Pathologists. Bibliographic references and other resources are included.

Eccentric Exercise in Treatment of Patellar Tendinopathy in High Level Basketball Players. A Randomized Clinical Trial.

Chronic patellar tendinopathy is a common pathology in sporting population. To date, there is no agreed upon protocol as election treatment. Eccentric exercises have been used with satisfactory outcomes (3). The purpose of this trial was to compare the effects of two eccentric exercise protocols.

Eccentric exercise in treatment of patellar tendinopathy in high level basketball players. A randomized clinical trial

Chronic patellar tendinopathy is a common pathology in sporting population. To date, there is no agreed upon protocol as election treatment. Eccentric exercises have been used with satisfactory outcomes.

Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+ cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+ cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+ cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+ cell ratio of 0.1. The CD8+ cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+ cell ratio of 0.25; the subject’s peripheral blood CD8+ cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+ cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+ cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+ cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.

Transgenic mice carrying a human mutant superoxide dismutase transgene develop neuronal cytoskeletal pathology resembling human amyotrophic lateral sclerosis lesions.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.

MicroRNAs in pathology and as therapeutic target in gene therapy?

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Clinical Streptococcus pneumoniae isolates induce differing CXCL8 responses from human nasopharyngeal epithelial cells which are reduced by liposomes.

BACKGROUND: Streptococcus pneumoniae causes several human diseases, including pneumonia and meningitis, in which pathology is associated with an excessive inflammatory response. A major inducer of this response is the cholesterol dependent pneumococcal toxin, pneumolysin. Here, we measured the amount of inflammatory cytokine CXCL8 (interleukin (IL)-8) by ELISA released by human nasopharyngeal epithelial (Detroit 562) cells as inflammatory response to a 24 h exposure to different pneumococcal strains. RESULTS: We found pneumolysin to be the major factor influencing the CXCL8 response. Cholesterol and sphingomyelin-containing liposomes designed to sequester pneumolysin were highly effective at reducing CXCL8 levels from epithelial cells exposed to different clinical pneumococcal isolates. These liposomes also reduced CXCL8 response from epithelial cells exposed to pneumolysin knock-out mutants of S. pneumoniae indicating that they also reduce the CXCL8-inducing effect of an unidentified pneumococcal virulence factor, in addition to pneumolysin. CONCLUSION: The results indicate the potential of liposomes in attenuating excessive inflammation as a future adjunctive treatment of pneumococcal diseases.

Bulletin of the Ayer Clinical Laboratory of the Pennsylvania Hospital.

No. 13-18 called also v.3