Bevacizumab and paclitaxel-carboplatin chemotherapy and secondary cytoreduction in recurrent, platinum-sensitive ovarian cancer (NRG Oncology/Gynecologic Oncology Group study GOG-0213): a multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: Platinum-based chemotherapy doublets are a standard of care for women with ovarian cancer recurring 6 months after completion of initial therapy. In this study, we aimed to explore the roles of secondary surgical cytoreduction and bevacizumab in this population, and report the results of the bevacizumab component here. METHODS: The multicentre, open-label, randomised phase 3 GOG-0213 trial was done in 67 predominantly academic centres in the USA (65 centres), Japan (one centre), and South Korea (one centre). Eligible patients were adult women (aged ≥18 years) with recurrent measurable or evaluable epithelial ovarian, primary peritoneal, or fallopian tube cancer, and a clinical complete response to primary platinum-based chemotherapy, who had been disease-free for at least 6 months following last infused cycle of platinum. Patients were randomly assigned (1:1) to standard chemotherapy (six 3-weekly cycles of paclitaxel [175 mg/m2 of body surface area] and carboplatin [area under the curve 5]) or the same chemotherapy regimen plus bevacizumab (15 mg/kg of bodyweight) every 3 weeks and continued as maintenance every 3 weeks until disease progression or unacceptable toxicity. Individuals who participated in both the bevacizumab objective and surgical objective (which is ongoing) were randomly assigned (1:1:1:1) to receive either of these two chemotherapy regimens with or without prior secondary cytoreductive surgery. Randomisation for the bevacizumab objective was stratified by treatment-free interval and participation in the surgical objective. The primary endpoint was overall survival, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00565851. FINDINGS: Between Dec 10, 2007, and Aug 26, 2011, 674 women were enrolled and randomly assigned to standard chemotherapy (n=337) or chemotherapy plus bevacizumab (n=377). Median follow-up at the end of the trial on Nov 5, 2014, was 49·6 months in each treatment group (IQR 41·5-62·2 for chemotherapy plus bevacizumab; IQR 40·8-59·3 for chemotherapy), at which point 415 patients had died (214 in the chemotherapy group and 201 in the chemotherapy plus bevacizumab group). Based on pretreatment stratification data, median overall survival in the chemotherapy plus bevacizumab group was 42·2 months (95% CI 37·7-46·2) versus 37·3 months (32·6-39·7) in the chemotherapy group (hazard ratio [HR] 0·829; 95% CI 0·683-1·005; p=0·056). We identified incorrect treatment-free interval stratification data for 45 (7%) patients (equally balanced between treatment groups); a sensitivity analysis of overall survival based on the audited treatment-free interval stratification data gave an adjusted HR of 0·823 (95% CI 0·680-0·996; p=0·0447). In the safety population (all patients who initiated treatment), 317 (96%) of 325 patients in the chemotherapy plus bevacizumab group had at least one grade 3 or worse adverse event compared with 282 (86%) of 332 in the chemotherapy group; the most frequently reported of these in the chemotherapy plus bevacizumab group compared with the chemotherapy group were hypertension (39 [12%] vs two [1%]), fatigue (27 [8%] vs eight [2%]), and proteinuria (27 [8%] vs none). Two (1%) treatment-related deaths occurred in the chemotherapy group (infection [n=1] and myelodysplastic syndrome [n=1]) compared with nine (3%) in the chemotherapy plus bevacizumab group (infection [n=1], febrile neutropenia [n=1], myelodysplastic syndrome [n=1], secondary malignancy [n=1]; deaths not classified with CTCAE terms: disease progression [n=3], sudden death [n=1], and not specified [n=1]). INTERPRETATION: The addition of bevacizumab to standard chemotherapy, followed by maintenance therapy until progression, improved the median overall survival in patients with platinum-sensitive recurrent ovarian cancer. Although the intention-to-treat analysis for overall survival was not significant, our sensitivity analysis based on corrected treatment-free interval stratification indicates that this strategy might be an important addition to the therapeutic armamentarium in these patients. FUNDING: National Cancer Institute and Genentech.

Somatic alterations in juvenile polyps from BMPR1A and SMAD4 mutation carriers.

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder predisposing to gastrointestinal hamartomatous polyps and cancer with a pathogenic SMAD4 or BMPR1A germline mutation (1st-hit) being identified in about 40-50% of patients. Little is known, however, about the occurrence and nature of somatic alterations (2nd-hit) in SMAD4-/BMPR1A-related juvenile polyps. In this study, we screened 25 polyps from three patients carrying either a pathogenic SMAD4 (c.1244-1247delACAG) or BMPR1A (c.583C>T; p.Gln195*) germline mutation for somatic alterations. The SMAD4-related polyps were also analyzed for SMAD4 protein expression by immunohistochemistry. Despite comprehensive screening for loss of heterozygosity (LOH), mutations in the coding sequence, chromosomal rearrangements, and promoter methylation, no somatic alterations could be identified in 14 SMAD4-related polyps. SMAD4 protein expression, however, was lost in 8 (57%) of 14 juvenile polyps with 6 showing concomitant loss in both, the epithelial and stromal, compartments. In the BMPR1A-related polyps, five out of nine (56%) displayed LOH. Further analysis of selected polyps revealed that LOH was gene copy number neutral and had occurred in the epithelial compartment. The heterogeneity of genetic mutations and protein expression levels indicates that different modes of gene inactivation can be operational in SMAD4- and BMPR1A-related polyp formation. Our observation, that about half of BMPR1A-related polyps displayed LOH, predominantly in the epithelial compartment, is compatible with BMPR1A acting as a tumour suppressor gene. Still, it remains to be determined whether juvenile polyp development generally requires loss of BMPR1A expression or, as observed in some SMAD4-related polyps, can occur despite normal protein expression.

Risk stratification of organ confined bladder cancer after radical cystectomy using cell cycle related biomarkers.

PURPOSE: We tested whether assessing the expression of cell cycle related proteins (p53, pRB, p21 and p27) could predict clinical outcomes after radical cystectomy in patients with organ confined urothelial carcinoma of the bladder. MATERIALS AND METHODS: Our study included a development cohort of 272 patients and an external testing cohort of 52 patients with chemotherapy naïve pT1-2N0M0 urothelial carcinoma of the bladder treated with radical cystectomy. Immunohistochemical staining of p53, p27, p21 and pRB was performed on the development cohort of 272 patients and the external testing cohort of 52 patients. RESULTS: Overall 260 (80.2%) patients had altered expression of at least 1 molecular marker and 105 (32.4%), 95 (29.3%), 44 (13.6%) and 16 (4.9%) had 1 to 4 altered molecular markers, respectively. Addition of the number of altered molecular markers increased the predictive accuracy of the base model for disease recurrence and cancer specific mortality by 15.6% and 14.8%, respectively (p <0.001). The base model included age, gender, pT1 vs pT2 stage, grade, number of lymph nodes removed, lymphovascular invasion and concomitant carcinoma in situ. The combination of molecular markers yielded a predictive accuracy superior to that of any single molecular marker. We developed nomograms for the prediction of recurrence-free and cancer specific survival. CONCLUSIONS: Assessment of the number of altered cell cycle regulatory proteins in the cystectomy specimen improves the prediction of urothelial carcinoma of the bladder recurrence and survival in patients with organ confined disease. A combination of multiple markers is needed to capture the complex biological behavior of urothelial carcinoma of the bladder.

Prognostic value of insulin-like growth factor II mRNA binding protein 3 in patients treated with radical prostatectomy.

UNLABELLED: Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Insulin-like growth factor II mRNA binding protein 3 (IMP3) is associated with poor outcomes in a variety of malignancies. The role of IMP3 in protate cancer remains poorly understood. IMP3 expression was associated with features of aggressive biology and aggressive prostate cancer recurrence after surgery. Although IMP3 is differentially expressed in patients with features of biologically aggressive prostate cancer, it does not have independent prognostic value in patients treated with RP. OBJECTIVE: To evaluate the association of insulin-like growth factor II mRNA binding protein 3 (IMP3) with pathological features and outcomes in patients treated with radical prostatectomy (RP). PATIENTS AND METHODS: Immunohistochemical staining for IMP3 was performed on archival tissue microarray specimens from 232 consecutive patients treated with RP for clinically localized disease. None of the patients received neoadjuvant or adjuvant radiation or hormone therapy. IMP3 expression was histologically categorized as normal or abnormal. Disease recurrence was classified as aggressive if metastases were present, post-recurrence prostate-specific antigen (PSA) doubling time was less than 10 months, or if the patients failed to respond to salvage local radiation therapy. RESULTS: The median follow-up was 69.8 months (interquartile range [IQR]: 40.1-99.5). IMP3 expression was abnormal in 42 (18.1%) of 232 patients. IMP3 expression was associated with extracapsular extension (P= 0.020), seminal vesicle invasion (P= 0.024), lymphovascular invasion (P= 0.036) and a high pathological Gleason score (P= 0.009). The 5-year PSA recurrence-free survival for IMP3-negative patients was 83% (standard error [SE]= 3) vs 67% (SE = 8) in IMP3-positive patients (log-rank test, P= 0.015). In a multivariable analysis that adjusted for the effects of surgical margins, extracapsular extension and seminal vesicle invasion, PSA (hazard ratio [HR]: 1.04, P= 0.013), lymph node metastasis (HR: 16.7, P < 0.001) and a high pathological Gleason score (HR 4.3, P= 0.008) were significantly associated with PSA recurrence-free survival, whereas IMP3 expression was not (P= 0.11). Similarly, IMP3 expression was only associated with aggressive recurrence (HR 3.2, P= 0.006). CONCLUSION: IMP3 expression is abnormal in approximately one-fifth of prostate cancers. Although IMP3 is differentially expressed in patients with features of biologically aggressive prostate cancer, it does not have an independent prognostic value in patients treated with RP.

Association of tumor-associated trypsin inhibitor (TATI) expression with molecular markers, pathologic features and clinical outcomes of urothelial carcinoma of the urinary bladder.

PURPOSES: To describe the differential tissue expression of tumor-associated trypsin inhibitor (TATI) in normal bladder urothelium, primary urothelial carcinoma of the bladder (UCB) and metastatic UCB and to assess the association of TATI expression with molecular markers commonly altered in UCB and clinical outcomes after radical cystectomy. METHODS: Slides from eight cystectomy patients without cancer, 191 radical cystectomy patients, 20 lymph nodes without metastasis and 40 lymph nodes with UCB were stained. Tissue expression of TATI, cyclin E1, cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, Caspase-3, Survivin and Cyclooxigenase-2 was measured in a tissue microarray. Cancer-specific and recurrence-free survival after radical cystectomy was recorded. RESULTS: TATI was expressed in 100% of patients without cancer, while 71% of radical cystectomy specimens and 90% of lymph node metastases exhibited decreased or no TATI expression. In radical cystectomy specimens, TATI expression decreased with advancing pathologic stage (P < 0.001) and lymphovascular invasion (P = 0.055). In univariate analyses, but not in multivariable Cox proportional hazard regression analyses, decreased TATI expression was associated with increased probability of tumor recurrence and cancer-specific mortality. Decreased TATI expression was correlated with altered expression of Cyclooxigenase-2 (P = 0.005), p21 (P = 0.035) and Ki-67 (P = 0.004). CONCLUSIONS: We found that normal urothelium expresses TATI and that TATI expression decreases with advancing tumor stage. While there was no prognostic benefit to TATI when adjusted for standard clinicopathologic features, it seems to play an important biologic role in UCB pathogenesis and invasion. Its association with markers involved in the cell cycle, proliferation and inflammation serves as hypothesis for molecular interactions.

Loss of androgen receptor expression is not associated with pathological stage, grade, gender or outcome in bladder cancer: a large multi-institutional study.

OBJECTIVE: • To investigate androgen receptor (AR) expression in a large series of patients with bladder cancer (BC) because data on a limited number of patients showed that loss of AR expression was associated with invasive BC. PATIENTS AND METHODS: • A total of 472 patients with urothelial bladder carcinoma (UBC) from two institutional centres (Toronto and Dallas) were analysed. Tissue microarrays comprising both non-muscle-invasive UBC (n= 167) and muscle-invasive UBC (n= 305) were accrued and immunohistochemical staining for AR was performed. • We used bright-field microscopy imaging coupled with advanced colour detection software to detect, classify and count stained cellular objects and manual scoring. • Results obtained in Dallas were blindly reviewed and validated in Toronto and samples randomly chosen were further analysed in Rochester, NY, USA. RESULTS: • The AR were positively expressed in 61/472 (12.9%) bladder tumours. No statistically significant difference in AR expression between men and women was observed. • Only 9.0% of non-muscle-invasive BC expressed the AR compared with 15.1% of muscle-invasive tumours (P= 0.059). The highest percentage of AR positivity (28.9% of cases) was found in T2 tumours. • There was no statistically significant difference in death from BC, time to death, or time to recurrence between AR-positive and AR-negative cases. CONCLUSION: • In contrast to previous reports, based on our large BC series, we did not observe a decrease in AR protein expression in bladder tumours with increased pathological stage. Our data do not suggest that loss of AR expression is gender-related nor is it associated with invasive BC.

Prospective validation of the clinical usefulness of reflex fluorescence in situ hybridization assay in patients with atypical cytology for the detection of urothelial carcinoma of the bladder.

PURPOSE: We previously prospectively evaluated the clinical usefulness of a fluorescence in situ hybridization assay for treating patients with atypical cytology results at risk for bladder cancer. This study is a prospective validation the usefulness of fluorescence in situ hybridization in this setting. MATERIALS AND METHODS: Between June 2007 and January 2009 every patient who underwent cystoscopy and cytology with atypical cytology underwent a reflex UroVysion test. A comprehensive review was then performed to evaluate clinical and pathological data on each patient. RESULTS: The study population comprised 108 patients with no history of cancer and 108 who underwent cystoscopy for cancer surveillance. In patients with cystoscopically visualized lesions UroVysion had a positive predictive value of 100% but there were false-negative results. In patients with equivocal cystoscopy and a history of cancer all 4 high grade tumors were detected and there were no false-negative findings. In patients with equivocal cystoscopy and no prior cancer the positive predictive value was 100% and there were no false-negative results. In patients with negative cystoscopy the UroVysion test detected all cancers but the positive predictive value was 10% and 29% in patients with and without a history of cancer, respectively. CONCLUSIONS: This prospective study of a reflex fluorescence in situ hybridization assay in patients with atypical cytology validates our previous findings. In patients with atypical cytology and obvious tumor on cystoscopy the assay was unnecessary but it was beneficial in those with equivocal or negative cystoscopy results. The fluorescence in situ hybridization assay identified all urothelial carcinoma tumors in patients with equivocal or negative cystoscopy. In patients with equivocal or negative cystoscopy and atypical cytology, a reflex fluorescence in situ hybridization assay may help avoid unnecessary evaluation while identifying those who would need further evaluation.

Association of angiogenesis related markers with bladder cancer outcomes and other molecular markers.

PURPOSE: We tested whether the altered immunohistochemical expression of angiogenesis related markers is associated with outcomes of patients with urothelial carcinoma of the bladder, and assessed the correlation of angiogenesis related markers with molecular markers commonly altered in urothelial bladder carcinoma. MATERIALS AND METHODS: Vascular endothelial growth factor, basic fibroblast growth factor and thrombospondin 1 expression data were collected, as were microvessel density data. Immunohistochemical staining was performed on specimens from 204 patients treated with radical cystectomy for urothelial carcinoma of the bladder. We also stained serial sections of the specimens for cyclin E1, cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, caspase-3, survivin and cyclooxygenase-2. We measured time to disease recurrence and cancer specific mortality, as well as the association with clinical and pathological features and other molecular markers. RESULTS: The altered expression of vascular endothelial growth factor (over expression), basic fibroblast growth factor (over expression) and thrombospondin 1 (decreased expression) was 86%, 79% and 63%, respectively. Median microvessel density was 20. All 4 markers were associated with established clinicopathological features of aggressive urothelial carcinoma of the bladder (such as stage, lymphovascular invasion and lymph node metastasis) and other molecular markers. On multivariable analyses that adjusted for standard pathological features basic fibroblast growth factor and thrombospondin 1 were independent predictors of disease recurrence (HR 3.6, p = 0.002 and HR 2.2, p = 0.001, respectively) and cancer specific mortality (HR 2.8, p = 0.02 and HR 2.3, p = 0.003, respectively). When all 4 markers were included in 1 model basic fibroblast growth factor and thrombospondin 1 retained their independent association with disease recurrence (HR 2.9, p = 0.014 and HR 1.8, p = 0.022, respectively) and only thrombospondin 1 was independently associated with cancer specific mortality (HR 1.9, p = 0.031). CONCLUSIONS: Angiogenesis related molecular markers are commonly altered in urothelial carcinoma of the bladder, making them a target for therapy. Down-regulation of thrombospondin 1 and up-regulation of basic fibroblast growth factor are independent predictors of clinical outcomes of patients with urothelial carcinoma of the bladder.

Combination of multiple molecular markers can improve prognostication in patients with locally advanced and lymph node positive bladder cancer.

PURPOSE: We tested whether the combination of 4 established cell cycle regulators (p53, pRB, p21 and p27) could improve the ability to predict clinical outcomes in a large multi-institutional collaboration of patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder. We also assessed whether the combination of molecular markers is superior to any individual biomarker. MATERIALS AND METHODS: The study comprised 692 patients with pT3-4N0 or pTany Npositive urothelial carcinoma of the bladder treated with radical cystectomy and bilateral lymphadenectomy (median followup 5.3 years). Scoring was performed using advanced cell imaging and color detection software. The base model incorporated patient age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of positive lymph nodes, concomitant carcinoma in situ and adjuvant chemotherapy. RESULTS: Individual molecular markers did not improve the predictive accuracy for disease recurrence and cancer specific mortality. Combination of all 4 molecular markers into number of altered molecular markers resulted in significantly higher predictive accuracy than any single biomarker (p <0.001). Moreover addition of number of altered molecular markers to the base model significantly improved the predictive accuracy for disease recurrence (3.9%, p <0.001) and cancer specific mortality (4.3%, p <0.001). Addition of number of altered molecular markers retained statistical significance for improving the prediction of clinical outcomes in the subgroup of patients with pT3N0 (280), pT4N0 (83) and pTany Npositive (329) disease (p <0.001). CONCLUSIONS: While the status of individual molecular markers does not add sufficient value to outcome prediction in patients with advanced urothelial carcinoma of the bladder, combinations of molecular markers may improve molecular staging, prognostication and possibly prediction of response to therapy.

Analysis of genetic copy number changes in cervical disease progression.

BACKGROUND: Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization. METHODS: The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity. RESULTS: The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells. CONCLUSIONS: The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression.

Human epidermal growth factor receptor 2 expression status provides independent prognostic information in patients with urothelial carcinoma of the urinary bladder.

OBJECTIVE: to test whether the expression of human epidermal growth factor receptor 2 (HER-2) is of prognostic value in a contemporary cohort of patients with urothelial carcinoma of the urinary bladder (UCB). PATIENTS AND METHODS: tissue microarrays of 198 patients were constructed and immunohistochemical stainings were performed on the primary tumours and on lymphatic nodal metastases. All patients were treated with radical cystectomy (RC) and regional lymphadenectomy for UCB. HER-2 expression was assessed using continuous HER-2 expression scores (ranging from 0.1 to 3.9) generated using an automated cellular imaging system. Scores of ≥ 1.0 in at least 10% of tumour cells were regarded as HER-2 positive. We correlated HER-2 scores with pathological and clinical variables, including disease recurrence and cancer-specific mortality. RESULTS: of 198 patients undergoing RC with lymphadenectomy, there was HER-2 positivity in 55 primary tumours (27.8%) compared with 44.2% of the evaluable positive lymph nodes (P < 0.001). HER-2 positivity was significantly associated with the presence of lymphovascular invasion (LVI; P= 0.026). With a median (range) follow-up of 35.4 (1.3-176.1) months, 101 patients (51.0%) had UCB recurrence and 82 patients (41.4%) died from the disease. In multivariable analyses that adjusted for the effects of pathological tumour stage, grade, LVI, lymph node metastasis and adjuvant chemotherapy, HER-2 positive patients were at increased risk for both UCB recurrence (hazard ratio [HR] 1.955, P= 0.003) and UCB-specific mortality (HR 2.066, P= 0.004) compared with patients with negative HER-2 expression. CONCLUSION: a positive HER-2 status is associated with aggressive UCB and provides independent prognostic information for UCB recurrence and mortality. Assessment of HER-2 status can be used to identify patients at high risk of disease progression who may benefit from adjuvant HER-2-targeted mono- or combined therapy after RC.

p53 expression in patients with advanced urothelial cancer of the urinary bladder.

OBJECTIVE: To test whether assessing p53 expression could improve the ability to predict disease recurrence and disease-specific survival in a multi-institutional cohort of patients with advanced urothelial carcinoma of the urinary bladder (UCB). PATIENTS AND METHODS: The study comprised 692 patients with pT3-4 N0 or pTany N+ UCB treated with radical cystectomy and lymphadenectomy. The predictive accuracy (PA) was quantified using the 200 bootstrap-corrected concordance index. The base model comprised age, gender, stage, grade, lymphovascular invasion, number of lymph nodes removed, number of lymph nodes positive, concomitant carcinoma in situ, and adjuvant chemotherapy. RESULTS: p53 expression was altered in 341 (49.3%) patients. In multivariable analyses, p53 expression was independently associated with disease recurrence (hazard ratio, 1.66; P < 0.001) and cancer-specific mortality (hazard ratio 1.65, P < 0.001). Overall, adding p53 did not significantly improve the PA of the base model (recurrence +0.7%, P = 0.085, and cancer-specific mortality +1.2%, P = 0.050). In the subgroups of pT3N0 (280) and pT4N0 (83) patients, p53 slightly improved the PA of the base model by a statistically significant degree (recurrence +1.7% and +3.6%, respectively; cancer-specific mortality +1.9% and +3.5%, respectively; all P < 0.001). In 329 patients with pTany N+ disease p53 status did not improve the PA of the base model. CONCLUSION: While assessing p53 expression has limited utility in patients with lymph node-positive UCB, it marginally improves prognostication in patients with advanced non-metastatic UCB. Integration of p53 into a panel of biomarkers might be necessary to capture a more accurate picture of the biological potential of advanced UCB.

Viable mice with compound mutations in the Wnt/Dvl pathway antagonists nkd1 and nkd2.

Gradients of Wnt/beta-catenin signaling coordinate development and physiological homeostasis in metazoan animals. Proper embryonic development of the fruit fly Drosophila melanogaster requires the Naked cuticle (Nkd) protein to attenuate a gradient of Wnt/beta-catenin signaling across each segmental anlage. Nkd inhibits Wnt signaling by binding the intracellular protein Dishevelled (Dsh). Mice and humans have two nkd homologs, nkd1 and nkd2, whose encoded proteins can bind Dsh homologs (the Dvl proteins) and inhibit Wnt signaling. To determine whether nkd genes are necessary for murine development, we replaced nkd exons that encode Dvl-binding sequences with IRES-lacZ/neomycin cassettes. Mutants homozygous for each nkd(lacZ) allele are viable with slightly reduced mean litter sizes. Surprisingly, double-knockout mice are viable, with subtle alterations in cranial bone morphology that are reminiscent of mutation in another Wnt/beta-catenin antagonist, axin2. Our data show that nkd function in the mouse is dispensable for embryonic development.

HMGA2 participates in transformation in human lung cancer.

Although previous studies have established a prominent role for HMGA1 (formerly HMG-I/Y) in aggressive human cancers, the role of HMGA2 (formerly HMGI-C) in malignant transformation has not been clearly defined. The HMGA gene family includes HMGA1, which encodes the HMGA1a and HMGA1b protein isoforms, and HMGA2, which encodes HMGA2. These chromatin-binding proteins function in transcriptional regulation and recent studies also suggest a role in cellular senescence. HMGA1 proteins also appear to participate in cell cycle regulation and malignant transformation, whereas HMGA2 has been implicated primarily in the pathogenesis of benign, mesenchymal tumors. Here, we show that overexpression of HMGA2 leads to a transformed phenotype in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA2 expression blocks the transformed phenotype in metastatic human non-small cell lung cancer cells. Moreover, we show that HMGA2 mRNA and protein are overexpressed in primary human lung cancers compared with normal tissue or indolent tumors. In addition, there is a statistically significant correlation between HMGA2 protein staining by immunohistochemical analysis and tumor grade (P < 0.001). Our results indicate that HMGA2 is an oncogene important in the pathogenesis of human lung cancer. Although additional studies with animal models are needed, these findings suggest that targeting HMGA2 could be therapeutically beneficial in lung cancer and other cancers characterized by increased HMGA2 expression.

Differential roles of telomere attrition in type I and II endometrial carcinogenesis.

Endometrial cancer has been generally categorized into two broad groups of tumors, type I (TI) and type II (TII), with distinct epidemiological/clinical features and genetic alterations. Because telomere attrition appears to trigger genomic instability in certain cancers, we explored the role of telomere dysfunction in endometrial cancer by analyzing telomeres and other markers of telomere status in both tumor types. We describe a new method, telomere chromogenic in situ hybridization, which permitted us to detect cells with short telomeres relative to control (stromal) cells within the same tissue section. Using this method, we found that both types of tumor cells had short telomeres. However, only TII tumors were significantly associated with critical telomere shortening in adjacent, morphologically normal epithelium, suggesting that telomere shortening contributes to the initiation of TII but not TI tumors. To explore this hypothesis, we analyzed mice with critically short telomeres and documented distinctive endometrial lesions that histologically resembled the in situ precursor of TII serous carcinomas; these lesions have not been observed previously in TI mouse models of endometrial cancer. Based on this and previous studies, we propose a model in which telomere attrition contributes to the initiation of TII and progression of TI endometrial cancers.

Predictive value of combined immunohistochemical markers in patients with pT1 urothelial carcinoma at radical cystectomy.

PURPOSE: pT1 urothelial carcinoma of the bladder is a potentially aggressive cancer diathesis with heterogeneous clinical behaviors. We tested whether the combination of immunohistochemical markers could risk stratify cases of pT1 urothelial carcinoma of the bladder at radical cystectomy. MATERIALS AND METHODS: p53, p21, pRB, p27, survivin and Ki-67 immunohistochemical staining was performed on representative urothelial carcinoma of the bladder specimens of 80 patients with pT1 urothelial carcinoma of the bladder treated with radical cystectomy and bilateral pelvic lymphadenectomy (median followup 61.6 months). RESULTS: p53 expression was altered in 25% of patients, p21 in 46%, pRB in 39%, p27 in 35%, survivin in 49% and Ki-67 in 34%. On multivariable analyses p53, p27 and Ki-67 were independently associated with urothelial carcinoma of the bladder recurrence (HR 3.66, p = 0.033; HR 3.76, p = 0.048 and HR 3.96, p = 0.021, respectively) and disease specific mortality (HR 5.25, p = 0.016; HR 3.68, p = 0.043 and HR 6.23, p = 0.009, respectively). The combination of these 3 biomarkers stratified cases into statistically significantly different risk groups for disease recurrence (p <0.001) and disease specific mortality (p <0.001). The addition of the number of altered markers increased the concordance indices of the base model that included grade, lymph node status, lymphovascular invasion and concomitant carcinoma in situ for disease recurrence and disease specific survival from 54.7% to 71.7% and from 64.3% to 77.5%, respectively. CONCLUSIONS: Assessment of p53, p27 and Ki-67 in urothelial carcinoma of the bladder specimens improves the prediction of recurrence-free and urothelial carcinoma of the bladder specific survival in patients with pT1 disease at radical cystectomy. These markers may help stratify the heterogeneous population of patients with pT1 disease into risk groups that can be used to guide clinical decision making regarding observation vs adjuvant therapy.

p53 predictive value for pT1-2 N0 disease at radical cystectomy.

PURPOSE: Approximately 15% to 30% of patients with pT1-2N0M0 urothelial carcinoma of the bladder experience disease progression despite radical cystectomy with curative intent. We determined whether p53 expression would improve the prediction of disease progression after radical cystectomy for pT1-2N0M0 UCB. MATERIALS AND METHODS: In a multi-institutional retrospective cohort we identified 324 patients with pT1-2N0M0 urothelial carcinoma of the bladder who underwent radical cystectomy. Analysis focused on a testing cohort of 272 patients and an external validation of 52. Competing risks regression models were used to test the association of variables with cancer specific mortality after accounting for nonbladder cancer caused mortality. RESULTS: In the testing cohort 91 patients (33.5%) had altered p53 expression (p53alt). On multivariate competing risks regression analysis altered p53 achieved independent status for predicting disease recurrence and cancer specific mortality (each p <0.001). Adding p53 increased the accuracy of multivariate competing risks regression models predicting recurrence and cancer specific mortality by 5.7% (62.0% vs 67.7%) and 5.4% (61.6% vs 67.0%), respectively. CONCLUSIONS: Alterations in p53 represent a highly promising marker of disease recurrence and cancer specific mortality after radical cystectomy for urothelial carcinoma of the bladder. Analysis confirmed previous findings and showed that considering p53 can result in substantial accuracy gains relative to the use of standard predictors. The value and the level of the current evidence clearly exceed previous proof of the independent predictor status of p53 for predicting recurrence and cancer specific mortality.

The high-mobility group A1 gene up-regulates cyclooxygenase 2 expression in uterine tumorigenesis.

Uterine cancer is the most common cancer of the female genital tract and is the fourth most frequent cause of cancer death in women in the U.S. Despite the high prevalence of uterine cancers, the molecular events that lead to neoplastic transformation in the uterus are poorly understood. Moreover, there are limited mouse models to study these malignancies. We generated transgenic mice with high-mobility group A1 gene (HMGA1a) expression targeted to uterine tissue and all female mice developed tumors by 9 months of age. Histopathologically, the tumors resemble human uterine adenosarcoma and are transplantable. To determine whether these findings are relevant to human disease, we evaluated primary human uterine neoplasms and found that HMGA1a mRNA and protein levels are increased in most high-grade neoplasms but not in normal uterine tissue, benign tumors, or most low-grade neoplasms. We also found that HMGA1a up-regulates cyclooxygenase 2 (COX-2) expression in transgenic tumors. Moreover, both HMGA1a and COX-2 expression are up-regulated in high-grade human leiomyosarcomas. Using chromatin immunoprecipitation, HMGA1a binds directly to the COX-2 promoter in human uterine cancer cells in vivo and activates its expression in transfection experiments. We also show that blocking either HMGA1a or COX-2 in high-grade human uterine cancer cells blocks anchorage-independent cell growth in methylcellulose. These findings show that HMGA1a functions as an oncogene when overexpressed in the uterus and contributes to the pathogenesis of human uterine cancer by activating COX-2 expression. Although a larger study is needed to confirm these results, HMGA1a may be a useful marker for aggressive human uterine cancers.

New markers of pancreatic cancer identified through differential gene expression analyses: claudin 18 and annexin A8.

BACKGROUND: New markers to distinguish benign reactive glands from infiltrating ductal adenocarcinoma of the pancreas are needed. DESIGN: The gene expression patterns of 24 surgically resected primary infiltrating ductal adenocarcinomas of the pancreas were compared with 18 non-neoplastic samples using the Affymetrix U133 Plus 2.0 Arrays and the Gene Logic GeneExpress Software System. Gene fragments from 4 genes (annexin A8, claudin 18, CXCL5, and S100 A2) were selected from the fragments found to be highly expressed in infiltrating adenocarcinomas when compared with normal tissues. The protein expression of these genes was examined using immunohistochemical labeling of tissue microarrays. RESULTS: Claudin 18 labeled infiltrating carcinomas in a membranous pattern. When compared with normal and reactive ducts, claudin 18 was overexpressed, at least focally, in 159 of 166 evaluable carcinomas (96%). Strong and diffuse claudin 18 overexpression was most often seen in well-differentiated carcinomas (P=0.02). Claudin 18 was overexpressed in 51 of 52 cases (98%) of pancreatic intraepithelial neoplasia. Annexin A8 was at least focally overexpressed in 149 of 154 evaluable infiltrating carcinomas (97%). S100 A2 was at least focally overexpressed in 118 of 154 evaluable infiltrating carcinomas (77%). Non-neoplastic glands also frequently expressed S100 A2 diminishing its potential diagnostic utility. Immunolabeling with antibodies directed against CXCL5 did not reveal any significant differences in protein expression between infiltrating adenocarcinomas and normal pancreatic ducts. CONCLUSIONS: Claudin 18 and annexin A8 are frequently highly overexpressed in infiltrating ductal adenocarcinomas when compared with normal reactive ducts, suggesting a role for these molecules in pancreatic ductal adenocarcinomas. Furthermore, these may serve as diagnostic markers, as screening tests and as therapeutic targets.

Prospective evaluation of the clinical usefulness of reflex fluorescence in situ hybridization assay in patients with atypical cytology for the detection of urothelial carcinoma of the bladder.

PURPOSE: The finding of atypical cytology is confusing for clinicians and it represents a management dilemma. We prospectively evaluated the clinical usefulness of fluorescence in situ hybridization assay for treating patients with atypical cytology who are at risk for bladder cancer. MATERIALS AND METHODS: Between May 2006 and June 2007 every patient who underwent cystoscopy and cytology with atypical or suspicious cytology underwent a reflex UroVysion test. A comprehensive review was then performed to evaluate clinical and pathological data on each patient. RESULTS: The population comprised 50 patients with no history of cancer and 70 who underwent cystoscopy for cancer surveillance. Fluorescence in situ hybridization assay was positive in all patients with cystoscopically visualized lesions whether they did or did not have a history of bladder cancer (positive predictive value 100%). In patients with equivocal cystoscopy and a history of cancer fluorescence in situ hybridization detected all 5 high grade tumors but it was false-negative for a low grade Ta tumor. In patients with equivocal cystoscopy and no prior cancer the positive predictive value was 50% and there was no false-negative assay result. In patients with negative cystoscopy and a history of cancer fluorescence in situ hybridization detected 3 cancers, including bladder carcinoma in situ in 2 and prostate carcinoma in situ in 1. In patients with no prior cancer and negative cystoscopy fluorescence in situ hybridization detected the only cancer (a high grade ureteral tumor) without yielding any false-negative results. CONCLUSIONS: This prospective evaluation of a reflex fluorescence in situ hybridization assay in patients with atypical cytology shows that the assay was unnecessary in patients with obvious tumors on cystoscopy but it was beneficial in patients with equivocal or negative cystoscopy. Fluorescence in situ hybridization assay identified all high grade cancers. It may help avoid unnecessary evaluation in patients with atypical cytology and equivocal or negative cystoscopy, while identifying those who would need further evaluation.