Intraosseous hibernoma: clinicopathologic and imaging analysis of 18 cases.

AIMS: Intraosseous hibernomas are rarely reported tumours with brown adipocytic differentiation of unknown aetiology, with only 38 cases documented in the literature. We sought to further characterise the clinicopathologic, imaging and molecular features of these tumours. METHODS AND RESULT: Eighteen cases were identified occurring in eight females and 10 males (median age = 65 years, range = 7-75). Imaging indication was cancer surveillance/staging in 11 patients and clinical concern for a metastasis was raised in 13 patients. The innominate bone (7), sacrum (5), mobile spine (4), humerus (1) and femur (1) were involved. Median tumour size was 1.5 cm (range = 0.8-3.8). Tumours were sclerotic (11), mixed sclerotic and lytic (4) or occult (1). Microscopically, tumours were composed of large polygonal cells with distinct cell membranes, finely vacuolated cytoplasm, central or paracentral small bland nuclei with prominent scalloping. Growth around trabecular bone was observed. Tumour cells were immunoreactive for S100 protein (15/15) and adipophilin (5/5), while negative for keratin AE1/AE3(/PCK26) (0/14) and brachyury (0/2). Chromosomal microarray analysis, performed on four cases, did not show clinically significant copy number variation across the genome or on 11q, the site of AIP and MEN1. CONCLUSION: Analysis of 18 cases of intraosseous hibernoma, to our knowledge, the largest series to date, revealed that these tumours are most often detected in the spine and pelvis of older adults. Tumours were generally small, sclerotic and frequently found incidentally and can raise concern for metastasis. Whether or not these tumours are related to soft tissue hibernomas is uncertain.

Integrative Clinical and Genomic Characterization of MTAP-deficient Metastatic Urothelial Cancer.

Deficiency of MTAP (MTAPdef) mainly occurs because of homozygous loss of chromosome 9p21, which is the most common copy-number loss in metastatic urothelial cancer (mUC). We characterized the clinical and genomic features of MTAPdef mUC in 193 patients treated at MD Anderson Cancer Center (MDACC) and 298 patients from the phase 2 IMvigor210 trial, which investigated atezolizumab in cisplatin-ineligible and platinum-refractory disease. In the MDACC cohort, visceral metastases were significantly more common for MTAPdef (n = 48) than for MTAP-proficient (MTAPprof; n = 145) patients (75% vs 55.2%; p = 0.02). MTAPdef was associated with poor prognosis (median overall survival [mOS] 12.3 vs 20.2 mo; p = 0.007) with an adjusted hazard ratio of 1.93 (95% confidence interval 1.35-2.98). Similarly, IMvigor210 patients with MTAPlo (n = 29) had a higher incidence of visceral metastases than those with MTAPhi tumors (n = 269; 86.2% vs 72.5%; p = 0.021) and worse prognosis (mOS 8.0 vs 11.3 mo; p = 0.042). Hyperplasia-associated genes were more frequently mutated in MTAPdef tumors (FGFR3: 31% vs 8%; PI3KCA: 31% vs 19%), while alterations in dysplasia-associated genes were less common in MTAPdef tumors (TP53: 41% vs 67%; RB1: 0% vs 16%). Our findings support a distinct biology in MTAPdef mUC that is associated with early visceral disease and worse prognosis. PATIENT SUMMARY: We investigated the outcomes for patients with the most common gene loss (MTAP gene) in metastatic cancer of the urinary tract. We found that this loss correlates with worse prognosis and a higher risk of metastasis in internal organs. There seems to be distinct tumor biology for urinary tract cancer with MTAP gene loss and this could be a potential target for treatment.

Longitudinal GFR trends after neoadjuvant chemotherapy prior to nephroureterectomy for upper tract urothelial carcinoma.

PURPOSE: Renal function dictates sequencing and eligibility for definitive therapy in upper tract urothelial carcinoma. We investigated longitudinal glomerular filtration rate (GFR) changes after neoadjuvant chemotherapy (NAC) and nephroureterectomy (RNU). MATERIALS AND METHODS: Patients treated with ≥3 cycles of chemotherapy prior to RNU for UTUC from 2000 to 2019 were included. GFR was calculated by CKD-Epi before chemotherapy, before RNU, 1 to 3 months, and 12 months post-RNU. Pathologic stage and overall survival were compared in those with stable GFR (+/-10% of baseline) to the rest of the cohort. RESULTS: One hundred and fifty-two patients received ≥3 cycles of NAC, with 121 (79%) receiving at least 1 cycle of cisplatin. Renal function dropped by mean of 22.3 ml/min/1.73 m2 from the beginning of chemotherapy to 1-year post-surgery. In patients receiving cisplatin, a mean decline of 26.2 ml/min/1.73 m2 was observed vs. 8.8 ml/min/1.73 m2 without cisplatin-based NAC (P < 0.01). GFR after RNU was unchanged between 3 and 12 months postoperatively. At 1 to 3 months after RNU, 19% of patients had GFR<30 ml/min/1.73m2. Improvement in GFR during NAC was associated with invasive final pathologic stage (P = 0.018) and worse overall survival (P = 0.049). CONCLUSIONS: In patients managed with NAC prior to RNU, renal function stabilizes at 1 to 3 months post-operatively and remains clinically similar for cisplatin or non-cisplatin-based therapy. Improvement in GFR during NAC was associated with higher pathologic stage and poorer survival, especially in those receiving non-cisplatin-based therapy, an observation that requires further investigation.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Estimation of tumor cell total mRNA expression in 15 cancer types predicts disease progression.

Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes.

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Secondary tumors of the bladder: A survival outcome study.

The urinary bladder may be involved by a variety of secondary tumors that originate from other organs. Bladder secondary tumors are rare and may be mistaken as bladder primary tumors because of their overlapping morphologic features. To avoid the diagnostic pitfalls, we analyzed the clinicopathologic features of bladder secondary tumors in a large cohort of patients. Our patient cohort consisted of 45 females and 38 males with a mean age of 58.7 ± 15.4 years (range 10-87 years). The tumors involved the bladder via direct extension from adjacent organs (n = 42) and distant metastasis (n = 41). In females, the majority of secondary tumors originated from the gynecologic tract (n = 25), and other common origins included the colon/rectum (n = 5) and breast (n = 4). In males, the most common origin was the prostate (n = 18), followed by the colon/rectum (n = 4) and kidney (n = 3). 75.9% of the secondary tumors were adenocarcinoma (n = 63), and other common tumor types included sarcoma (n = 6), squamous cell carcinoma (n = 5), melanoma (n = 4), and neuroendocrine carcinoma (n = 3). 67.5% of patients (n = 56) died of the disease with a median overall survival of 23 months from the time of secondary involvement of the bladder. Patients with secondary tumors via direct extension had a median survival time of 20 months, which was not significantly different from that for patients with secondary involvement via distant metastasis (24 months) (p = 0.83). Median survival in cases with prostate primary was 20 months as compared to 23 months for all other tumor types (p = 0.68). The majority of secondary tumors are composed of adenocarcinoma, which highlights the importance of differentiating primary from secondary involvement in bladder adenocarcinoma. Regardless of the origin, bladder secondary tumors are associated with a poor prognosis.

Total Tibial Allograft Reconstruction for Adamantinoma: A Case Report With 2-Year Follow-up.

CASE: A 47-year-old woman with adamantinoma of the entire left tibia and distal fibula underwent resection and reconstruction using a total tibia allograft-prosthetic composite with rotating hinged knee replacement and ankle fusion. She is ambulating without tumor recurrence with 2-year follow-up. CONCLUSION: This case report offers a unique reconstruction option for extensive tibia bone primary malignancy. To our knowledge, this is the longest survival for total tibia allograft prosthetic composite reconstruction.

Time-to-progression after front-line fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy for chronic lymphocytic leukaemia: a retrospective, multicohort study.

BACKGROUND: Fludarabine, cyclophosphamide, and rituximab (FCR) has become a gold-standard chemoimmunotherapy regimen for patients with chronic lymphocytic leukaemia. However, the question remains of how to treat treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia. We therefore aimed to develop and validate a gene expression signature to identify which of these patients are likely to achieve durable remissions with FCR chemoimmunotherapy. METHODS: We did a retrospective cohort study in two cohorts of treatment-naive patients (aged ≥18 years) with chronic lymphocytic leukaemia. The discovery and training cohort consisted of peripheral blood samples collected from patients treated at the University of Texas MD Anderson Cancer Center (Houston, TX, USA), who fulfilled the diagnostic criteria of the International Workshop on Chronic Lymphocytic Leukemia, had received at least three cycles of FCR chemoimmunotherapy, and had been treated between Oct 10, 2000, and Oct 26, 2006 (ie, the MDACC cohort). We did transcriptional profiling on samples obtained from the MDACC cohort to identify genes associated with time to progression. We did univariate Cox proportional hazards analyses and used significant genes to cluster IGHV-unmutated samples into two groups (intermediate prognosis and unfavourable prognosis). After using cross-validation to assess robustness, we applied the Lasso method to standardise the gene expression values to find a minimum gene signature. We validated this signature in an external cohort of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia enrolled on the CLL8 trial of the German Chronic Lymphocytic Leukaemia Study Group who were treated between July 21, 2003, and April 4, 2006 (ie, the CLL8 cohort). FINDINGS: The MDACC cohort consisted of 101 patients and the CLL8 cohort consisted of 109 patients. Using the MDACC cohort, we identified and developed a 17-gene expression signature that distinguished IGHV-unmutated patients who were likely to achieve a long-term remission following front-line FCR chemoimmunotherapy from those who might benefit from alternative front-line regimens (hazard ratio 3·83, 95% CI 1·94-7·59; p<0·0001). We validated this gene signature in the CLL8 cohort; patients with an unfavourable prognosis versus those with an intermediate prognosis had a cause-specific hazard ratio of 1·90 (95% CI 1·18-3·06; p=0·008). Median time to progression was 39 months (IQR 22-69) for those with an unfavourable prognosis compared with 59 months (28-84) for those with an intermediate prognosis. INTERPRETATION: We have developed a robust, reproducible 17-gene signature that identifies a subset of treatment-naive patients with IGHV-unmutated chronic lymphocytic leukaemia who might substantially benefit from treatment with FCR chemoimmunotherapy. We recommend testing the value of this gene signature in a prospective study that compares FCR treatment with newer alternative therapies as part of a randomised clinical trial. FUNDING: Chronic Lymphocytic Leukaemia Global Research Foundation and the National Institutes of Health/National Cancer Institute.

Outcomes of nonmetastatic micropapillary variant upper tract urothelial carcinoma.

PURPOSE: Micropapillary variant upper tract urothelial cancer (MP-UTUC) is a rare malignancy with little known regarding its clinical course and/or optimal treatment. In this case series, we describe patient characteristics, surgical treatment, oncologic outcomes, and response to perioperative chemotherapy. MATERIALS AND METHODS: We conducted a review to identify patients with MP-UTUC treated at our center between January 1994 and October 2017. Clinicopathologic data was obtained. Descriptive statistics, Kaplan-Meier analysis, Cox proportional hazards, and nearest neighbor matching were used to examine the cohort. RESULTS: Eighteen, (4.3%) of 416 patients were found to have MP-UTUC at our institution over a 23-year period. The majority of patients had ≥pT3 disease at the time of extirpative surgery (13/18, 72%) and one was identified as MP-UTUC prior to surgery. Seven patients received neoadjuvant chemotherapy and six patients received adjuvant chemotherapy. Median overall, cancer specific, and recurrence free survival were 3.29, 3.29, and 1.69 years, respectively for MP-UTUC. There was no survival difference between conventional UTUC and MP-UTUC when matched for age, stage, grade, lymphovascular invasion, and margins (HR 1.18, P = 0.567). No MP-UTUC patients receiving neoadjuvant and adjuvant chemotherapy had apparent pathologic down staging, and of those receiving adjuvant chemotherapy two-thirds died of disease within 2 years. CONCLUSIONS: MP-UTUC is a rare, and in most cases aggressive malignancy that commonly presents as locally advanced disease. In this case series, MP-UTUC does not appear to respond to perioperative chemotherapy as neoadjuvant and adjuvant chemotherapy did not result in apparent pathologic down staging and the majority of those receiving adjuvant chemotherapy died from MP-UTUC.

Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

Impact of High-risk Features and Effect of Neoadjuvant Chemotherapy in Urothelial Cancer Patients with Invasion into the Lamina Propria on Transurethral Resection in the Absence of Deep Muscle Invasion.

BACKGROUND: High-risk non-muscle-invasive bladder cancer (NMIBC) that invades into the lamina propria is frequently understaged and is associated with a risk of lymph node metastasis and death. OBJECTIVE: To identify high-risk features (HRFs) for NMIBC that may identify patients with poorer prognosis who may benefit from neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC). DESIGN, SETTING, AND PARTICIPANTS: We performed a single-center retrospective review of patients who underwent RC for NMIBC with invasion into the lamina propria between 1995 and 2013. HRFs included hydronephrosis, abnormal examination under anesthesia, lymphovascular invasion, or variant histology. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Pathology at RC, and overall (OS) and disease-specific (DSS) survival were evaluated and analyzed by Fisher's exact test, Student t test, Cox proportional hazards regression analysis, and the Kaplan-Meier method. RESULTS AND LIMITATIONS: We identified 336 patients with a median follow-up of 130 mo. Of these, 159 (47%) had no HRF, 140 (41.5%) had one HRF, and 37 (11%) had ≥2 HRFs. At RC, patients with ≥2 HRFs had a significantly higher rate of pathologic T stage upstaging and lymph node metastasis (p<0.05). Median OS was 139 mo for those with no HRF, 127 mo for those with one HRF, and 56 mo for those with ≥2 HRF (p=0.0057). HRFs are also associated with a decreased DSS (p=0.0009). Patients with ≥2 HRFs (11/37) who received NAC showed improved OS (21% vs 55% 5-yr OS, p=0.0353) and trended toward an improvement in DSS (25% vs 56% 5-yr OS, p=0.0716) compared with RC alone. CONCLUSIONS: The presence of ≥2 HRFs in NMIBC invading the lamina propria is associated with worse pathology at RC and a significant decrease in OS and DSS. NAC appears to provide benefit for these patients. Limitations include retrospective design and limited sample size. PATIENT SUMMARY: The presence of high-risk features in urothelial cancer with invasion into the lamina propria has a worse prognosis that may be mitigated by neoadjuvant chemotherapy.

Comprehensive Genomic Characterization of Upper Tract Urothelial Carcinoma.

BACKGROUND: Upper urinary tract urothelial cancer (UTUC) may have unique etiologic and genomic factors compared to bladder cancer. OBJECTIVE: To characterize the genomic landscape of UTUC and provide insights into its biology using comprehensive integrated genomic analyses. DESIGN, SETTING, AND PARTICIPANTS: We collected 31 untreated snap-frozen UTUC samples from two institutions and carried out whole-exome sequencing (WES) of DNA, RNA sequencing (RNAseq), and protein analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Adjusting for batch effects, consensus mutation calls from independent pipelines identified DNA mutations, gene expression clusters using unsupervised consensus hierarchical clustering (UCHC), and protein expression levels that were correlated with relevant clinical variables, The Cancer Genome Atlas, and other published data. RESULTS AND LIMITATIONS: WES identified mutations in FGFR3 (74.1%; 92% low-grade, 60% high-grade), KMT2D (44.4%), PIK3CA (25.9%), and TP53 (22.2%). APOBEC and CpG were the most common mutational signatures. UCHC of RNAseq data segregated samples into four molecular subtypes with the following characteristics. Cluster 1: no PIK3CA mutations, nonsmokers, high-grade

Clinical risk stratification in patients with surgically resectable micropapillary bladder cancer.

OBJECTIVE: To analyse survival in patients with clinically localised, surgically resectable micropapillary bladder cancer (MPBC) undergoing radical cystectomy (RC) with and without neoadjuvant chemotherapy (NAC) and develop risk strata based on outcome data. PATIENTS AND METHODS: A review of our database identified 103 patients with surgically resectable (≤cT4acN0 cM0) MPBC who underwent RC. Survival estimates were calculated using Kaplan-Meier method and compared using log-rank tests. Classification and regression tree (CART) analysis was performed to identify risk groups for survival. RESULTS: For the entire cohort, estimated 5-year overall survival and disease-specific survival (DSS) rates were 52% and 58%, respectively. CART analysis identified three risk subgroups: low-risk: cT1, no hydronephrosis; high-risk: ≥cT2, no hydronephrosis; and highest-risk: cTany with tumour-associated hydronephrosis. The 5-year DSS for the low-, high-, and highest-risk groups were 92%, 51%, and 17%, respectively (P < 0.001). Patients down-staged at RC

Functional CAR models for large spatially correlated functional datasets.

We develop a functional conditional autoregressive (CAR) model for spatially correlated data for which functions are collected on areal units of a lattice. Our model performs functional response regression while accounting for spatial correlations with potentially nonseparable and nonstationary covariance structure, in both the space and functional domains. We show theoretically that our construction leads to a CAR model at each functional location, with spatial covariance parameters varying and borrowing strength across the functional domain. Using basis transformation strategies, the nonseparable spatial-functional model is computationally scalable to enormous functional datasets, generalizable to different basis functions, and can be used on functions defined on higher dimensional domains such as images. Through simulation studies, we demonstrate that accounting for the spatial correlation in our modeling leads to improved functional regression performance. Applied to a high-throughput spatially correlated copy number dataset, the model identifies genetic markers not identified by comparable methods that ignore spatial correlations.

RGS6 is an essential tumor suppressor that prevents bladder carcinogenesis by promoting p53 activation and DNMT1 downregulation.

Urinary bladder cancer (UBC) is largely caused by exposure to toxic chemicals including those in cigarette smoke (i.e. BBN). An activating SNP in RGS6 is associated with a pronounced reduction in UBC risk, especially among smokers. However, the mechanism underlying this reduction remains unknown. Here we demonstrate that RGS6 is robustly expressed in human urothelium, where urothelial cell carcinoma originates, and is downregulated in human UBC. Utilizing RGS6-/- mice we interrogated a possible role for RGS6 as a tumor suppressor using the BBN-induced bladder carcinogenesis model that closely recapitulates human disease. As in humans, RGS6 is robustly expressed in mouse urothelium. RGS6 loss dramatically accelerates BBN-induced bladder carcinogenesis, with RGS6-/- mice consistently displaying more advanced pathological lesions than RGS6+/+ mice. Furthermore, BBN treatment promotes urothelial RGS6 mRNA and protein downregulation. RGS6 loss impairs p53 activation and promotes aberrant accumulation of oncogenic protein DNMT1 in urothelium. Tumor suppressor RASSF1A, a DNMT1-regulated gene, is also silenced, likely via methylation of its promoter during BBN exposure. We hypothesize that this BBN-induced RGS6 loss represents a critical hit in UBC as it irrevocably impairs the anti-proliferative actions of the ATM/p53 and RASSF1A pathways. Consistent with these findings, RGS6-/- mice treated with CP-31398, a p53-stablizing agent, and/or 5-Aza, a DNMT1 inhibitor, are protected from BBN-induced tumorigenesis. Together, our data identify RGS6 as a master tumor suppressor modulating two critical signaling pathways that are often dysregulated in UBC; therefore, RGS6 represents a potential novel biomarker for UBC diagnosis/prognosis and an appealing new target in its treatment.

Comparison between whole mount tissue preparations and virtual tissue microarray samples for measuring Ki-67 and apoptosis indices in human bladder cancer: A cross-sectional study.

Recent tissue microarray (TMA)-based studies have shown that cell proliferation- and apoptosis-related biomarkers are associated with clinical outcomes in patients with bladder urothelial carcinoma. However, little is known about the differences in these biomarker measurements between whole mount tissue preparations and TMAs. This study aimed to elucidate the discrepancy in the measurements of Ki-67 indices (KIs) and apoptosis indices (AIs) between whole mount tissue preparations and TMAs of bladder urothelial carcinoma samples.Whole mount tissue preparations for Ki-67 immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling were made from 30 patients who underwent transurethral resection of bladder urothelial carcinoma. Digital microscopy-assisted virtual TMAs, consisting of 3 small round areas (1 or 0.6 mm in diameter), were generated from the same whole mount tissue preparations. The measurement results in highly reactive areas of biomarkers were compared between the whole mount tissue preparation- and the TMA-based methods. Bland-Altman plot analysis, regression analysis, and Kendall τ were performed to investigate differences in the measurement results, systematic biases, and correlations between biomarkers.Although the Bland-Altman plot analysis demonstrated that almost all the plots were within the limits of agreement, fixed biases were detected in the 1- and 0.6-mm TMAs for the KI (0.181 and 0.222, respectively) and the AI (0.055 and 0.063, respectively). Proportional biases were also detected in the 1- and 0.6-mm TMAs for the AI (P < 0.001 and P < 0.001, respectively). Furthermore, positive correlations between KIs and AIs were observed in whole mount tissue preparations (r = 0.260, P = 0.044) and in the 1 mm TMAs (r = 0.375, P = 0.004); however, no such correlation was observed in the 0.6 mm TMAs.Our study suggests that the measurement results for certain biomarkers of bladder urothelial carcinoma obtained from TMA-based samples can be susceptible to systematic bias, and the lack of correlation between biomarkers cannot be avoided as it is in whole mount tissue preparations. Virtual TMAs can help identify systematic bias and establish a better sampling strategy prior to performing high-throughput TMAs for biomarker studies.

Next-generation sequencing identifies high frequency of mutations in potentially clinically actionable genes in sebaceous carcinoma.

Sebaceous carcinoma (SC) is a rare but aggressive malignancy with frequent recurrence and metastases. Surgery is the mainstay of therapy, but effective systemic therapies are lacking because the molecular alterations driving SC remain poorly understood. To identify these, we performed whole-exome next-generation sequencing of 409 cancer-associated genes on 27 SCs (18 primary/locally recurrent ocular, 5 paired metastatic ocular, and 4 primary extraocular) from 20 patients. In ocular SC, we identified 139 non-synonymous somatic mutations (median/lesion 3; range 0-23). Twenty-five of 139 mutations (18%) occurred in potentially clinically actionable genes in 6 of 16 patients. The most common mutations were mutations in TP53 (n = 9), RB1 (n = 6), PIK3CA (n = 2), PTEN (n = 2), ERBB2 (n = 2), and NF1 (n = 2). TP53 and RB1 mutations were restricted to ocular SC and correlated with aberrant TP53 and RB protein expression. Systematic pathway analyses demonstrated convergence of these mutations to activation of the PI3K signalling cascade, and PI3K pathway activation was confirmed in tumours with PTEN and/or PIK3CA mutations. Considerable inter-tumoural heterogeneity was observed between paired primary and metastatic ocular SCs. In primary extraocular SC, we identified 77 non-synonymous somatic mutations (median/lesion 22.5; range 3-29). This overall higher mutational load was attributed to a microsatellite instability phenotype in three of four patients and somatically acquired mutations in mismatch repair genes in two of four patients. Eighteen of 77 mutations (23%) were in potentially clinically actionable genes in three of four patients, including BTK, FGFR2, PDGFRB, HRAS, and NF1 mutations. Identification of potentially clinically actionable mutations in 9 of 20 SC patients (45%) underscores the importance of next-generation sequencing to expand the spectrum of genotype-matched targeted therapies. Frequent activation of PI3K signalling pathways provides a strong rationale for application of mTOR inhibitors in the management of this disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Integrating Murine and Clinical Trials with Cabozantinib to Understand Roles of MET and VEGFR2 as Targets for Growth Inhibition of Prostate Cancer.

PURPOSE: We performed parallel investigations in cabozantinib-treated patients in a phase II trial and simultaneously in patient-derived xenograft (PDX) models to better understand the roles of MET and VEGFR2 as targets for prostate cancer therapy. EXPERIMENTAL DESIGN: In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR2, bone turnover, angiogenesis, and resistance were examined. RESULTS: In responsive patients and PDX, islets of viable pMET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice nor did it affect cabozantinib-induced growth inhibition but did lead to induction of FGFR1. Inhibition of VEGFR2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR2-negative vessel. Reduction of bone turnover was observed in both cohorts. CONCLUSIONS: Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic castrate-resistant prostate cancer (CRPC), but inhibition of VEGFR2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents.