Coexistent intraurothelial carcinoma and muscle-invasive urothelial carcinoma of the bladder: clonality and somatic down-regulation of DNA mismatch repair

<p>Muscle-invasive urothelial carcinomas are heterogeneous neoplasms for which the clonal relationship with low-grade urothelial dysplasia and carcinomas in situ remains unknown, and both monoclonal and field change models have been proposed. Low-grade dysplasia (18) and carcinoma in situ (12) associated with muscle-invasive urothelial carcinoma were microdissected and topographically analyzed (intraepithelial and invasive superficial and deep to muscularis mucosa) for methylation pattern of androgen receptor alleles, TP53, RB1, WT1, and NF1 microsatellite analysis to assess clonal identity; MLH1 and MSH2 sequencing/immunostaining. Appropriate controls were run. Carcinoma in situ (100%) and invasive urothelial carcinoma (100%) revealed monoclonal patterns, whereas low-grade dysplasia was preferentially polyclonal (80%). Carcinoma in situ showed aneuploid DNA content and more abnormal microsatellites than the corresponding invasive compartments, opposite to low-grade dysplasia. Absent MLH1 protein expression with no gene mutations were identified in carcinoma in situ and nodular-trabecular urothelial carcinoma with high microsatellite abnormalities. Somatic mismatch repair protein down-regulation and the accumulation of tumor suppressor gene microsatellite abnormalities contribute to a molecular evolution for monoclonal carcinoma in situ divergent from coexistent muscle-invasive urothelial carcinoma. Low-grade dysplasia is however unlikely connected with this molecular progression.</p

Kinetic profiles by topographic compartments in muscle-invasive transitional cell carcinomas of the bladder: role of TP53 and NF1 genes

<p>We evaluated 71 muscle-invasive transitional cell carcinomas (TCCs) of the bladder by tumor compartments. Kinetic parameters included mitotic figure counting, Ki-67 index, proliferation rate (DNA slide cytometry), and apoptotic index (in situ end labeling [ISEL] of fragmented DNA using digoxigenin-labeled deoxyuridine triphosphate and Escherichia coli DNA polymerase [Klenow fragment]). At least 50 high-power fields per compartment were screened from the same tumor areas; results are expressed as percentage of positive neoplastic cells. Mean and SD were compared by tumor compartment. DNA was extracted from microdissected samples (superficial and deep) and used for microsatellite analysis of TP53 and NF1 by polymerase chain reaction-denaturing gradient gel electrophoresis. Significantly higher marker scores were revealed in the superficial compartment than in the deep compartment. An ISEL index of less than 1% was revealed in 63% (45/71) of superficial compartments and 86% (61/71) of deep compartments. Isolated NF1 alterations were observed mainly in superficial compartments, whereas isolated TP53 abnormalities were present in deep compartments. Lower proliferation and down-regulation of apoptosis define kinetically the deep compartment of muscle-invasive TCC of the bladder and correlate with the topographic heterogeneity, NF1-defective in superficial compartments and TP53-defective in deep compartments.</p

Molecular evolution and intratumor heterogeneity by topographic compartments in muscle-invasive transitional cell carcinoma of the urinary bladder

<p>Superficial transitional cell carcinomas (TCC) of the urinary bladder have been shown to be monoclonal. However, no combined study of clonality and tumor suppressor genes (TSG) is available to date for muscle-invasive TCC. Forty-four muscle-invasive TCC of the urinary bladder selected from women were included in this study. Tumor cells located above and below the muscularis mucosa zone were systematically microdissected and used for DNA extraction. Hha-I digested and undigested samples were used to study the methylation pattern of androgen receptor alleles and undigested samples were used for microsatellite analysis of TSG (TP53, RB1, WT1, and NF1). Both loss of heterozygosity (LOH) and single nucleotide polymorphism (SNP) analyses were performed using optimized denaturing gradient gel electrophoresis. The expression of p53, pRB, and p21WAF1 was assessed by immunohistochemistry. Appropriate controls were run in every case. All except two TCC showed a monoclonal pattern with the same allele inactivated in both compartments. Microsatellite analysis of TSG revealed the same LOH/SNP pattern in both tumor compartments in 30 cases (involving more than 1 TSG locus in 8) and genetic heterogeneity in 14 cases. From the latter group, 9 cases expressed more genetic changes in the deep compartment (involving TP53 gene in all cases, WT1 gene in 2, and NF1 in 1), whereas in 4 cases the superficial compartment showed more genetic changes (three involving NF1 and one involving both RB and TP53). No statistical difference in the immunoexpression was detected, although it tended to be higher in the superficial compartment than in the deep compartment. These concordant data in polymorphic DNA regions indicate that bladder-muscle-invasive TCC are monoclonal proliferations with homogeneous tumor cell selection. Heterogeneous tumor cell selection by topography defined two different genetic compartments: superficial, NF1-defective, and deep, TP53-defective. No differences in the immunohistochemical expression were observed, precluding a more extensive clinical application.</p

ARE INTRAUROTHELIAL LESIONS ASSOCIATED WITH MUSCLE-INVASIVE TRANSITIONAL CELL CARCINOMAS TRUE PRECURSORS?

<p>Background: The clonal relationship between low-grade urothelial dysplasia (LGUD) and carcinomas in situ (CIS) with muscle- invasive transitional cell carcinoma (TCC) remains unknown.<br>Methods: LGUD (18) and CIS (12) from 72 patients with muscle-invasive TCC (coexistent lesions in 5 patients) were selected and microdissected. DNA was extracted from dysplastic cells (LGUD and CIS) and invasive tumor cells located above (superficial) and below (deep) the muscularis mucosa and used to analyze the methylation pattern of androgen receptor alleles. The microsatellite patterns of TP53, RBI, WT1, and NFI were studied by PCR-denaturing gradient gel electrophoresis. The same areas were systematically s t u d i a p r the expression of Ki67 and cell cycle regulators (p53, p21 , pRBl), nuclear DNA content, and in situ end labeling. Appropriate controls were run in each case.<br>Results: Monoclonal patterns were revealed in CIS (6, loo%), invasive TCC (13, 100%), and LGUD (2, 20%), whereas polyclonal patterns were observed in LGUD only (8, 80%). CIS showed aneuploid DNA content and more microsatellite loci altered than the corresponding invasive compartments, always involving TP53 loci and expressing abnormal p53. In contrast, LGUD revealed diploid DNA content and microsatellite abnormalities in only 2 cases, one monoclonal (RBI) and one polyclonal (WT1 and NF1). Opposite kinetic patterns were observed for CIS (higher Ki-67 index, lower ISEL index) and LGUD (lower Ki-67 index, higher ISEL index).<br>Conclusions: These genetic findings suggest that CIS evolution is independent of muscle-invasive ?CC and that secondary CIS is not the precursor lesion of coexistent TCC. LGUD should not be closely connected with this molecular progression. The kinetic patterns would contribute to the accumulation of genetic abnormalities in CIS but not in LGU.</p

Poor Prognosis and a Distinctive Genetic Profile Define Muscle- Invasive Transitional Cell Carcinoma of the Bladder with Single-File Infiltration Pattern

<p>Background: Histologic patterns have been demonstrated prognostically useful in transitional cell carcinomas (TCC) of the bladder, especially for the distinction between superficial and invasive neoplasms. No information is available on the prognostic significance and genetic profile of invasive patterns in muscle-invasive TCC.<br>Methods: Seventy-twomuscle-invasiveTCC of the bladder were histologically evaluated regarding a pondered histologic grade (low vs. high), and MIB-1 index. The infiltrationpattern was assessed in the deep compartment and classified according to the predominant pattern as ‘solid’ (macronodules or diffuse infiltration effacing the muscle fibers) or ‘single-file’ (S-F = single-cell tumor infiltration dissecting the muscle and inducing a dense stromal reaction). DNA was extracted from at least 2 samples per tumor and used to analyze the microsatellite pattern of TP53, RB1, WT1, and NF1 by PCR - denaturing gradient gel electrophoresis. The data were statistically evaluated by ANOVA and Fisher’s exact test.<br>Results: Solid TCC revealed significantly higher MIB- 1 index than SF infiltrating TCC, tended to show higher proportion of low-grade TCC, and longer survival.<br>The genetic profile demonstrated significant differences only for RB1 -and NF1 abnormalities (present in solid TCC), and more genetic alterations in solid TCC.<br>Conclusions: Solid infiltration in muscle-invasive TCC correlates with higher proliferation, higher incidence of RB1 and NF1 abnormalities,and longer survival.</p

HIGH PROLIFERATION AND NORMAL APOPTOSIS ARE THE KINETIC HALLMARKS OF CUTANEOUS MERKEL CELL CARCINOMAS

<p>Background: Merkel cell carcinomas (MCCs) are unusual cutaneous neoplasms that reveal both epithelial and neuroendocrine differentiation. No detailed analysis of cell kinetics (proliferation and apoptosis) by topographic compartments is available to date. Methods: All MCCs were required to express at least two epithelial and two neural markers from a panel including cytokeratin cocktail AEl -AE3, cytokeratin 20, synaptophysin, chromogranin A, neurofilament protein, and neuron-specific enolase, along with consistent ultrastructural findings. We selected 19 MCCs to evaluate mitotic figure (MF) counting, Ki-67 index, and apoptosis index based on the in situ end labeling (ISEL) of fragmented DNA using digoxigenin-labeled dUTP and Escherichia coli DNA polymerase I (Klenow fragment). At least 50 high-power fields were screened per topographic compartment (superficial or papillary dermis, and deep or reticular dermis), recording average and standard deviation for each variable. Variables were statistically compared in each tumor compartment using analysis of variance and Student t-test (significant if P<0.05.<br>Results: MCCs revealed high cell density (over 425 cell/HPF) and no statistical differences for theproliferation-markers by topographic compartments. Apoptosis showed lower values in the deep compartment, but only significant for the standard deviation of ISEL index (P=0.0074).<br>Conclusions: Homogeneously distributed high proliferation defines MCCs. Apoptosis follows the distribution pattern of proliferation in superficial compartments, whereas in deep compartments the apoptosis distribution is less variable and independent from proliferation.</p

Validation of the Collaborative Outcomes study on Health and Functioning during Infection Times (COH-FIT) questionnaire for adults

Background: The Collaborative Outcome study on Health and Functioning during Infection Times (COH-FIT; www.coh-fit.com) is an anonymous and global online survey measuring health and functioning during the COVID-19 pandemic. The aim of this study was to test concurrently the validity of COH-FIT items and the internal validity of the co-primary outcome, a composite psychopathology “P-score”. Methods: The COH-FIT survey has been translated into 30 languages (two blind forward-translations, consensus, one independent English back-translation, final harmonization). To measure mental health, 1–4 items (“COH-FIT items”) were extracted from validated questionnaires (e.g. Patient Health Questionnaire 9). COH-FIT items measured anxiety, depressive, post-traumatic, obsessive-compulsive, bipolar and psychotic symptoms, as well as stress, sleep and concentration. COH-FIT Items which correlated r ≥ 0.5 with validated companion questionnaires, were initially retained. A P-score factor structure was then identified from these items using exploratory factor analysis (EFA) and confirmatory factor analyses (CFA) on data split into training and validation sets. Consistency of results across languages, gender and age was assessed. Results: From >150,000 adult responses by May 6th, 2022, a subset of 22,456 completed both COH-FIT items and validated questionnaires. Concurrent validity was consistently demonstrated across different languages for COH-FIT items. CFA confirmed EFA results of five first-order factors (anxiety, depression, post-traumatic, psychotic, psychophysiologic symptoms) and revealed a single second-order factor P-score, with high internal reliability (ω = 0.95). Factor structure was consistent across age and sex. Conclusions: COH-FIT is a valid instrument to globally measure mental health during infection times. The P-score is a valid measure of multidimensional mental health