Wiking Månsson

A Molecular Taxonomy for Urothelial Carcinoma

Purpose: Even though urothelial cancer is the fourth most common tumor type among males, progress in treatment has been scarce. A problem in day-to-day clinical practice is that precise assessment of individual tumors is still fairly uncertain; consequently efforts have been undertaken to complement tumor evaluation with molecular biomarkers. An extension of this approach would be to base tumor classification primarily on molecular features. Here, we present a molecular taxonomy for urothelial carcinoma based on integrated genomics. Experimental Design: We use gene expression profiles from 308 tumor cases to define five major urothelial carcinoma subtypes: urobasal A, genomically unstable, urobasal B, squamous cell carcinoma like, and an infiltrated class of tumors. Tumor subtypes were validated in three independent publically available data sets. The expression of 11 key genes was validated at the protein level by immunohistochemistry. Results: The subtypes show distinct clinical outcomes and differ with respect to expression of cell-cycle genes, receptor tyrosine kinases particularly FGFR3, ERBB2, and EGFR, cytokeratins, and cell adhesion genes, as well as with respect to FGFR3, PIK3CA, and TP53 mutation frequency. The molecular subtypes cut across pathologic classification, and class-defining gene signatures show coordinated expression irrespective of pathologic stage and grade, suggesting the molecular phenotypes as intrinsic properties of the tumors. Available data indicate that susceptibility to specific drugs is more likely to be associated with the molecular stratification than with pathologic classification. Conclusions: We anticipate that the molecular taxonomy will be useful in future clinical investigations. Clin Cancer Res; 18(12); 3377–86. ©2012 AACR.

MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31.

We analyzed 34 cases of urothelial carcinomas by miRNA, mRNA and genomic profiling. Unsupervised hierarchical clustering using expression information for 300 miRNAs produced 3 major clusters of tumors corresponding to Ta, T1 and T2‐T3 tumors, respectively. A subsequent SAM analysis identified 51 miRNAs that discriminated the 3 pathological subtypes. A score based on the expression levels of the 51 miRNAs, identified muscle invasive tumors with high precision and sensitivity. MiRNAs showing high expression in muscle invasive tumors included miR‐222 and miR‐125b and in Ta tumors miR‐10a. A miRNA signature for FGFR3 mutated cases was also identified with miR‐7 as an important member. MiR‐31, located in 9p21, was found to be homozygously deleted in 3 cases and miR‐452 and miR‐452* were shown to be over expressed in node positive tumors. In addition, these latter miRNAs were shown to be excellent prognostic markers for death by disease as outcome. The presented data shows that pathological subtypes of urothelial carcinoma show distinct miRNA gene expression signatures.

Combined Gene Expression and Genomic Profiling Define Two Intrinsic Molecular Subtypes of Urothelial Carcinoma and Gene Signatures for Molecular Grading and Outcome

In the present investigation, we sought to refine the classification of urothelial carcinoma by combining information on gene expression, genomic, and gene mutation levels. For these purposes, we performed gene expression analysis of 144 carcinomas, and whole genome array-CGH analysis and mutation analyses of FGFR3, PIK3CA, KRAS, HRAS, NRAS, TP53, CDKN2A, and TSC1 in 103 of these cases. Hierarchical cluster analysis identified two intrinsic molecular subtypes, MS1 and MS2, which were validated and defined by the same set of genes in three independent bladder cancer data sets. The two subtypes differed with respect to gene expression and mutation profiles, as well as with the level of genomic instability. The data show that genomic instability was the most distinguishing genomic feature of MS2 tumors, and that this trait was not dependent on TP53/MDM2 alterations. By combining molecular and pathologic data, it was possible to distinguish two molecular subtypes of T(a) and T(1) tumors, respectively. In addition, we define gene signatures validated in two independent data sets that classify urothelial carcinoma into low-grade (G(1)/G(2)) and high-grade (G(3)) tumors as well as non-muscle and muscle-invasive tumors with high precisions and sensitivities, suggesting molecular grading as a relevant complement to standard pathologic grading. We also present a gene expression signature with independent prognostic effect on metastasis and disease-specific survival. We conclude that the combination of molecular and histopathologic classification systems might provide a strong improvement for bladder cancer classification and produce new insights into the development of this tumor type.

ICUD-EAU International Consultation on Bladder Cancer 2012: Urinary diversion.

CONTEXT A summary of the 2nd International Consultation on Bladder Cancer recommendations on the reconstructive options after radical cystectomy (RC), their outcomes, and their complications. OBJECTIVE To review the literature regarding indications, surgical details, postoperative care, complications, functional outcomes, as well as quality-of-life measures of patients with different forms of urinary diversion (UD). EVIDENCE ACQUISITION An English-language literature review of data published between 1970 and 2012 on patients with UD following RC for bladder cancer was undertaken. No randomized controlled studies comparing conduit diversion with neobladder or continent cutaneous diversion have been performed. Consequently, almost all studies used in this report are of level 3 evidence. Therefore, the recommendations given here are grade C only, meaning expert opinion delivered without a formal analysis. EVIDENCE SYNTHESIS Indications and patient selection criteria have significantly changed over the past 2 decades. Renal function impairment is primarily caused by obstruction. Complications such as stone formation, urine outflow, and obstruction at any level must be recognized early and treated. In patients with orthotopic bladder substitution, daytime and nocturnal continence is achieved in 85-90% and 60-80%, respectively. Continence is inferior in elderly patients with orthotopic reconstruction. Urinary retention remains significant in female patients, ranging from 7% to 50%. CONCLUSIONS RC and subsequent UD have been assessed as the most difficult surgical procedure in urology. Significant disparity on how the surgical complications were reported makes it impossible to compare postoperative morbidity results. Complications rates overall following RC and UD are significant, and when strict reporting criteria are incorporated, they are much higher than previously published. Fortunately, most complications are minor (Clavien grade 1 or 2). Complications can occur up to 20 yr after surgery, emphasizing the need for lifelong monitoring. Evidence suggests an association between surgical volume and outcome in RC; the challenge of optimum care for elderly patients with comorbidities is best mastered at high-volume hospitals by high-volume surgeons. Preoperative patient information, patient selection, surgical techniques, and careful postoperative follow-up are the cornerstones to achieve good long-term results.

Should All Patients with Non–Muscle-Invasive Bladder Cancer Receive Early Intravesical Chemotherapy after Transurethral Resection? The Results of a Prospective Randomised Multicentre Study

BACKGROUND To decrease recurrences in non-muscle-invasive bladder cancer (NMIBC), the European Association of Urology (EAU) guidelines recommend immediate, intravesical chemotherapy after transurethral resection (TUR) for all patients with Ta/T1 tumours. OBJECTIVE To study the benefits of a single, early, intravesical instillation of epirubicin after TUR in patients with low- to intermediate-risk NMIBC. DESIGN, SETTING, AND PARTICIPANTS In this prospective randomised multicentre trial, 305 patients with primary as well as recurrent low- to intermediate-risk (Ta/T1, G1/G2) tumours were enrolled between 1997 and 2004. Patients were randomly allocated to receive 80 mg of epirubicin in 50 ml of saline intravesically within 24 h of TUR or no further treatment after TUR. MEASUREMENTS The primary end point was time to first recurrence. RESULTS AND LIMITATIONS A total of 219 patients remained for analysis after exclusions. The median follow-up time was 3.9 yr. During the study period, 62% (63 of 102) of the patients in the epirubicin group and 77% (90 of 117) in the control group experienced recurrence (p=0.016). In a multivariate model, the hazard ratio (HR) for recurrence was 0.56 (p=0.002) for early instillation of epirubicin versus no treatment. In a subgroup analysis, the treatment had a profound recurrence-reducing effect on patients with primary, solitary tumours, whereas it provided no benefits in patients with recurrent or multiple tumours. Furthermore, patients with a modified European Organisation for Research and Treatment of Cancer (EORTC) risk score of 0-2 with and without single instillation had recurrence rates of 41% and 69%, respectively (p=0.003), whereas the corresponding rates for those with a risk score of > or = 3 were 81% and 85%, respectively (p=0.35). CONCLUSIONS A single, early instillation of epirubicin after TUR for NMIBC reduces the likelihood of tumour recurrence; however, the benefit seems to be minimal in patients at intermediate or high risk of recurrence. Future trials will determine the value of early instillation in addition to serial instillations in NMIBC.

The quality of life in men after radical cystectomy with a continent cutaneous diversion or orthotopic bladder substitution: is there a difference?

Objective  To compare the quality of life (QoL) in men after radical cystectomy who had either a continent cutaneous diversion or orthotopic bladder substitution.

Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma.

Similar to other malignancies, urothelial carcinoma (UC) is characterized by specific recurrent chromosomal aberrations and gene mutations. However, the interconnection between specific genomic alterations, and how patterns of chromosomal alterations adhere to different molecular subgroups of UC, is less clear. We applied tiling resolution array CGH to 146 cases of UC and identified a number of regions harboring recurrent focal genomic amplifications and deletions. Several potential oncogenes were included in the amplified regions, including known oncogenes like E2F3, CCND1, and CCNE1, as well as new candidate genes, such as SETDB1 (1q21), and BCL2L1 (20q11). We next combined genome profiling with global gene expression, gene mutation, and protein expression data and identified two major genomic circuits operating in urothelial carcinoma. The first circuit was characterized by FGFR3 alterations, overexpression of CCND1, and 9q and CDKN2A deletions. The second circuit was defined by E3F3 amplifications and RB1 deletions, as well as gains of 5p, deletions at PTEN and 2q36, 16q, 20q, and elevated CDKN2A levels. TP53/MDM2 alterations were common for advanced tumors within the two circuits. Our data also suggest a possible RAS/RAF circuit. The tumors with worst prognosis showed a gene expression profile that indicated a keratinized phenotype. Taken together, our integrative approach revealed at least two separate networks of genomic alterations linked to the molecular diversity seen in UC, and that these circuits may reflect distinct pathways of tumor development.

Intraoperative sentinel node detection improves nodal staging in invasive bladder cancer.

PURPOSE We evaluated intraoperative SN detection in patients with invasive bladder cancer during radical cystectomy in conjunction with extended lymphadenectomy. MATERIALS AND METHODS A total of 75 patients with invasive bladder cancer underwent radical cystectomy with extended lymphadenectomy. SNs were identified by preoperative lymphoscintigraphy, intraoperative dynamic lymphoscintigraphy and blue dye detection. An isotope (70 MBq (99m)Tc-nanocolloid) and Patent Blue(R) blue dye were injected peritumorally via a cystoscope. Excised lymph nodes were examined ex vivo using a handheld gamma probe. Identified SNs were evaluated by extended serial sectioning, hematoxylin and eosin staining, and immunohistochemistry. RESULTS At lymphadenectomy an average of 40 nodes (range 8 to 67) were removed. Of 75 patients 32 (43%) were lymph node positive, of whom 13 (41%) had all lymph node metastases located only outside of the obturator spaces. An SN was identified in 65 of 75 patients (87%). In 7 patients an SN was recognized when the nodal basins were assessed with the gamma probe after lymphadenectomy and cystectomy. Of the 32 lymph node positive cases 26 (81%) had a positive (metastatic) SN. Thus, the false-negative rate was 6 of 32 cases (19%). Five false-negative cases had macrometastases and/or perivesical metastases. In 9 patients (14%) the SN contained micrometastases (less than 2 mm), in 5 of whom the micrometastasis was the only metastatic deposit. CONCLUSIONS SN detection is feasible in invasive bladder cancer, although the false- negative rate was 19% in this study. Extended serial sectioning and immunohistochemistry revealed micrometastases in SNs in 9 patients and radio guided surgery after the completion of lymphadenectomy identified SNs in an additional 7. We believe that the technique that we used in this study improved nodal staging in these 16 of 65 patients (25%).

Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

We studied if bladder cancers respond to HAMLET (human α‐lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native α‐lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.

A Systematic Study of Gene Mutations in Urothelial Carcinoma; Inactivating Mutations in TSC2 and PIK3R1.

Background Urothelial carcinoma (UC) is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations. Methodology/Principal Findings We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%. Conclusions/Significance Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.