Development and Independent Validation of a Prognostic Gene Expression Signature Based on RB1, PTEN, and TP53 in Metastatic Hormone-sensitive Prostate Cancer Patients.

BACKGROUND: Androgen deprivation therapy (ADT) with docetaxel (D) and/or antiandrogen receptor therapies (ARTs) are the standard therapies in metastatic hormone-sensitive prostate cancer (mHSPC). Alterations in the tumor suppressor genes (TSGs) RB1, PTEN, and TP53 are associated with an aggressive evolution and treatment resistance in castration-resistant prostate cancer (CRPC). OBJECTIVE: To study the clinical implications of TSG mRNA expression in mHSPC patients. DESIGN, SETTING, AND PARTICIPANTS: This is a multicenter retrospective biomarker study in mHSPC patients. TSGlow status was defined when two or more out of the three TSGs presented low RNA expression by nCounter in formalin-fixed paraffin-embedded samples and TSGwt for the remaining cases. The microarray data from the CHAARTED trial were analyzed as an independent validation cohort. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Molecular data were correlated with CRPC-free survival (CRPC-FS) and overall survival (OS) by the Kaplan-Meier method and multivariate Cox analysis. RESULTS AND LIMITATIONS: A total of 226 patients were included, of whom 218 were eligible: 93 were treated with ADT and 125 with ADT + D; 75.7% presented de novo stage IV and 67.9% high-volume disease. TSGlow (19.2%) was independently correlated with shorter CRPC-FS (hazard ratio [HR] 1.8, p = 0.002) and OS (HR 2, p = 0.002). In the CHAARTED trial, TSGlow was independently correlated with lower CRPC-FS (HR 2.2, p = 0.02); no differences in clinical outcomes according to treatment were observed in TSGlow patients, while a significant benefit was observed for ADT + D in the TSGwt group for CRPC-FS (HR 0.4, p < 0.001) and OS (HR 0.4, p = 0.001). However, no interaction was observed between TSG signature and treatment in either series. Study limitations are the retrospective design, small sample size, and lack of inclusion of patients treated with ADT + ART. CONCLUSIONS: TSGlow expression correlates with adverse outcomes in patients with mHSPC. The investigation of new therapeutic strategies in these patients is warranted. PATIENT SUMMARY: The low RNA expression of tumor suppressor genes in the tumors is correlated with adverse outcomes in patients with metastatic hormone-sensitive prostate cancer.

Tumor-Agnostic Circulating Tumor DNA Testing for Monitoring Muscle-Invasive Bladder Cancer.

Circulating tumor DNA (ctDNA) has recently emerged as a real-time prognostic and predictive biomarker for monitoring cancer patients. Here, we aimed to ascertain whether tumor-agnostic ctDNA testing would be a feasible strategy to monitor disease progression and therapeutic response in muscle-invasive bladder cancer (MIBC) patients after radical cystectomy (RC). Forty-two MIBC patients who underwent RC were prospectively included. Blood samples from these patients were collected at different follow-up time points. Two specific mutations (TERT c.1-124C>T and ATM c.1236-2A>T) were analyzed in the patients' plasma samples by droplet digital PCR to determine their ctDNA status. During a median follow-up of 21 months, 24% of patients progressed in a median of six months. ctDNA status was identified as a prognostic biomarker of tumor progression before RC and 4 and 12 months later (HR 6.774, HR 3.673, and HR 30.865, respectively; p < 0.05). Lastly, dynamic changes in ctDNA status between baseline and four months later were significantly associated with patient outcomes (p = 0.045). In conclusion, longitudinal ctDNA analysis using a tumor-agnostic approach is a potential tool for monitoring MIBC patients after RC. The implementation of this testing in a clinical setting could improve disease management and patients' outcomes.

Assessment of aggressive bladder cancer mutations in plasma cell-free DNA.

Background and aims: The spatial and temporal genetic heterogeneity of bladder cancer (BC) makes challenging to find specific drivers of metastatic disease, thus preventing to determine those BC patients at high risk of tumor progression. Our aim was to identify DNA mutations providing aggressive behavior to bladder tumors and analyze them in patients' cell-free DNA (cfDNA) during their follow-up after radical cystectomy (RC) in order to monitor tumor evolution. Methods: Six BC patients who underwent RC and presented disease progression during their follow-up were included. Next-generation sequencing was used to determine somatic mutations in several primary tumor and metastatic specimens from each patient. Shared DNA mutations between primary bladder tumor and metastatic sites were identified in cfDNA samples through droplet digital PCR. Results: Besides BC genetic heterogeneity, specific mutations in at least one of these genes -TERT, ATM, RB1, and FGFR3- were found in primary tumors and their metastases in all patients. These mutations were also identified in the patients' cfDNA at different follow-up time points. Additionally, the dynamic changes of these mutations in cfDNA allowed us to determine tumor evolution in response to treatment. Conclusion: The analysis of BC mutations associated with poor prognosis in plasma cfDNA could be a valuable tool to monitor tumor evolution, thus improving the clinical management of BC patients.

P53 in Penile Squamous Cell Carcinoma: A Pattern-Based Immunohistochemical Framework with Molecular Correlation.

p53 immunohistochemistry (IHC) has been proposed as a surrogate for TP53 mutations in penile squamous cell carcinomas (PSCC). We aimed to evaluate the performance of a pattern-based evaluation of p53 IHC in PSCC. Human papilloma virus (HPV) DNA testing, p16 and p53 IHC, and whole exome sequencing were performed in a series of 40 PSCC. p53 IHC was evaluated following a pattern-based framework and conventional p53 IHC evaluation. Out of 40 PSCC, 12 (30.0%) were HPV-associated, and 28 (70.0%) were HPV-independent. The agreement between the p53 IHC pattern-based evaluation and TP53 mutational status was almost perfect (k = 0.85). The sensitivity and accuracy of the pattern-based framework for identifying TP53 mutations were 95.5% and 92.5%, respectively, which were higher than the values of conventional p53 IHC interpretation (54.5% and 70.0%, respectively), whereas the specificity was the same (88.9%). In conclusions, the pattern-based framework improves the accuracy of detecting TP53 mutations in PSCC compared to the classical p53 IHC evaluation.

Transcriptional Profile Associated with Clinical Outcomes in Metastatic Hormone-Sensitive Prostate Cancer Treated with Androgen Deprivation and Docetaxel.

(1) Background: Androgen deprivation therapy (ADT) and docetaxel (DX) combination is a standard therapy for metastatic hormone-sensitive prostate cancer (mHSPC) patients. (2) Methods: We investigate if tumor transcriptomic analysis predicts mHSPC evolution in a multicenter retrospective biomarker study. A customized panel of 184 genes was tested in mRNA from tumor samples by the nCounter platform in 125 mHSPC patients treated with ADT+DX. Gene expression was correlated with castration-resistant prostate cancer-free survival (CRPC-FS) and overall survival (OS). (3) Results: High expression of androgen receptor (AR) signature was independently associated with longer CRPC-FS (hazard ratio (HR) 0.6, 95% confidence interval (CI) 0.3-0.9; p = 0.015), high expression of estrogen receptor (ESR) signature with longer CRPC-FS (HR 0.6, 95% CI 0.4-0.9; p = 0.019) and OS (HR 0.5, 95% CI 0.2-0.9, p = 0.024), and lower expression of tumor suppressor genes (TSG) (RB1, PTEN and TP53) with shorter OS (HR 2, 95% CI 1-3.8; p = 0.044). ARV7 expression was independently associated with shorter CRPC-FS (HR 1.5, 95% CI 1.1-2.1, p = 0.008) and OS (HR 1.8, 95% CI 1.2-2.6, p = 0.004), high ESR2 was associated with longer OS (HR 0.5, 95% CI 0.2-1, p = 0.048) and low expression of RB1 was independently associated with shorter OS (HR 1.9, 95% CI 1.1-3.2, p = 0.014). (4) Conclusions: AR, ESR, and TSG expression signatures, as well as ARV7, RB1, and ESR2 expression, have a prognostic value in mHSPC patients treated with ADT+DX.

Cell-Free DNA as a Prognostic Biomarker for Monitoring Muscle-Invasive Bladder Cancer.

Cell-free DNA (cfDNA) has recently emerged as a real-time biomarker for diagnosis, monitoring and prediction of therapy response in tumoral disease. Here, we evaluated cfDNA as a prognostic biomarker for monitoring muscle-invasive bladder cancer (MIBC) patients at different follow-up time points. Blood samples from 37 MIBC patients who underwent radical cystectomy (RC) were collected at cystectomy and 1, 4, 12 and 24 months later. Plasma cfDNA amount and fragmentation patterns were determined. Four mutations were analyzed in cfDNA to detect circulating tumor DNA (ctDNA) during patient follow-up. During a median follow-up of 36 months, 46% of patients progressed; median time to progression was 10 months. cfDNA levels and ctDNA status four months after RC were identified as independent prognostic biomarkers of tumor progression (HR 5.290; p = 0.033) and cancer-specific survival (HR 4.199; p = 0.038), respectively. Furthermore, ctDNA clearance four months after RC was significantly associated with patients' clinical outcomes. In conclusion, cfDNA levels and ctDNA status four months after RC have prognostic implications in MIBC patients. In addition, cfDNA monitoring is useful to predict patient outcomes after RC. cfDNA analysis in the clinical setting could greatly improve MIBC patient management.

Histone H4 acetylation is dysregulated in active seminiferous tubules adjacent to testicular tumours.

STUDY QUESTION: Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours? SUMMARY ANSWER: A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis. WHAT IS KNOWN ALREADY: Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients. STUDY DESIGN, SIZE, DURATION: A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation. LIMITATIONS, REASONS FOR CAUTION: Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the 'Ministerio de Economía y Competividad (Spain)' (fondos FEDER 'una manera de hacer Europa', PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Short-term effectiveness and tolerability of carbon dioxide laser for anal high-grade squamous intraepithelial lesions in individuals living with HIV.

BACKGROUND: Ablative treatment of intra-anal high-grade squamous intraepithelial lesions (HSIL) reduces the risk of progression to anal squamous cell carcinoma. Our objective was to assess the short-term effectiveness and tolerability of the carbon dioxide laser for treating intra-anal HSIL in patients at high risk of anal cancer. METHODS: This is an exploratory, pilot, single-arm, clinical trial of treatment response for anal HSIL in people living with HIV diagnosed with ≤3 not previously treated HSILs. Individuals were treated with one carbon dioxide laser treatment session. Clinical assessment by high resolution anoscopy and systematic recording of adverse events was performed. RESULTS: Fifty-two patients with 72 HSILs were included. Response to treatment was assessed in 48 (92.3%) patients; in the per-protocol population analysis, complete, partial, and no response was seen in 50% (n = 24), 20.8% (n = 10) and 29.1% (n = 14), respectively. Being older than 40 years and having a CD4 T-cell count lower than 200 cells/µL at diagnosis of HSIL were significantly associated with a poor response to treatment. Data on adverse events was recorded for 49 patients and 69.4% (n=34) reported no symptoms after the procedure. CONCLUSIONS: Carbon dioxide laser ablation is a promising and well tolerated treatment for intra-anal HSIL.

Response factors associated with electrocautery treatment of intra-anal high-grade squamous intraepithelial lesions in a population of HIV-positive men who have sex with men.

BACKGROUND: Ablative treatment of anal high-grade squamous intraepithelial lesions (HSIL) reduces the risk of progression to anal squamous cell carcinoma. OBJECTIVES: To identify factors that influence the response to treatment of anal HSIL by electrocautery ablation (ECA) in a population of HIV-positive men who have sex with men (MSM). DESIGN: Retrospective study of ECA treatment response in a prospectively followed anal dysplasia cohort. HIV-positive MSM diagnosed with anal HSIL were included. Demographic and HIV data were recorded. Response to treatment was assessed by biopsy after at least 18 months of follow-up. RESULTS: One hundred and twenty-eight HSILs in 91 men were included in this study. The overall response rate at 18 months was 70.3%. The number of electrocautery sessions required (2 ECA sessions vs 1: adjusted odds ratio [aOR] = 0.36 (95%CI 0.13-1.01); >=3 sessions vs 1: aOR = 0.10 (95%CI 0.04-0.29); p < 0.001]) and the history of previous HPV-related anal pathology (previous anal lesions vs no previous lesions AOR = 2.83 (95%CI 1.14-7.02), p = 0.024) were independently associated with response at 18 months. No serious adverse events were reported. CONCLUSIONS: Consideration should be given to alternative therapies in patients with unresolved HSIL after 1 ECA treatment.

Effect of vascularized periosteum on revitalization of massive bone isografts: An experimental study in a rabbit model.

INTRODUCTION: In the last years, limb salvage has become the gold standard treatment over amputation. Today, 90% of extremity osteogenic sarcomas can be treated with limb salvage surgery. However, these reconstructions are not exempt from complications. Massive allografts have been associated to high risk of nonunion (12-57%), fracture (7-30%) and infection (5-21%). Association of vascularized periosteum flap to a massive bone allograft (MBA) has shown to halve the average time of allograft union in clinical series, even compared to vascularized fibular flap. Creeping substitution process has been reported in massive allograft when periosteum flap was associated. However, we have little data about whether it results into allograft revitalization. We hypothesize that the association of a periosteum flap to a bone isograft promotes isograft revitalization, defined as the colonization of the devitalized bone by new-form vessels and viable osteocytes, turning it vital. MATERIALS AND METHODS: Forty-four New Zealand white male rabbits underwent a 10 mm segmental radial bone defect. In 24 rabbits the bone excision included the periosteum (controls); in 20 rabbits (periosteum group) bone excision was performed carefully detaching periosteum in order to preserve it. Cryopreserved bone isograft from another rabbit was trimmed and placed to the defect gap and was fixed with a retrograde intramedullar 0.6 mm Kirschner wire. Rabbits were randomized and distributed in 3 subgroups depending on the follow-up (control group: 5 rabbits in 5-week follow up group, 8 rabbits in 10-week follow-up group, 7 rabbits in 20-week follow-up group; periosteum group: 5 rabbits in 5-week follow up group, 7 rabbits in 10-week follow-up group, 7 rabbits in 20-week follow-up group). Fluoroscopic images of rabbit forelimb were taken after sacrifice to address union. Each specimen was blindly evaluated in optical microscope (magnification, ×4) after hematoxylin and eosin staining to qualitative record: presence of new vessels and osteocytes in bone graft lacunae (yes/no) to address revitalization, presence of callus (yes/no) and woven bone and cartilage tissue area (mm2 ) to address remodeling (osteoclast resorption of old bone and substitution by osteoblastic new bone formation). RESULTS: No isograft revitalization occurred in any group, but it was observed bone graft resorption and substitution by new-formed bone in periosteum group. This phenomenon was accelerated in 5-week periosteum group (control group: 49.5 ± 9.6 mm2 vs. periosteum group: 34.9 ± 10.4 mm2 ; p = .07). Remodeled lamellar bone was observed in both 20-week groups (control group: 6.1 ± 6.3 mm2 vs. periosteum group: 5.8 ± 3.0 mm2 , p = .67). Periosteum group showed complete integration and graft substitution, whereas devitalized osteons were still observed in 20-week controls. All periosteum group samples showed radiographic union through a bone callus, whereas controls showed nonunion in eight specimens (Union rate: control group 60% vs. periosteum group 100%, p = .003). CONCLUSIONS: Association of vascularized periosteum to a massive bone isograft has shown to accelerate bone graft substitution into a newly formed bone, thus, no bone graft revitalization occurs.

Cell Plasticity-Related Phenotypes and Taxanes Resistance in Castration-Resistant Prostate Cancer.

The prostatic tumor cells plasticity is involved in resistance to hormone-therapy, allowing these cells to survive despite androgen receptor inhibition. However, its role in taxanes resistance has not been fully established. Gene expression of plasticity-related phenotypes such as epithelial-mesenchymal transition (EMT), stem cell-like and neuroendocrine (NE) phenotypes was studied in vitro, in silico, in circulating tumor cells (CTCs) (N=22) and in tumor samples (N=117) from taxanes-treated metastatic castration-resistant prostate cancer (mCRPC) patients. Docetaxel (D)-resistant cells presented a more pronounced EMT phenotype than cabazitaxel (CZ)-resistant cells. In silico analysis revealed ESRP1 down-regulation in taxane-exposed mCRPC samples. Cell plasticity-related changes occurred in CTCs after taxanes treatment. Tumor EMT phenotype was associated with lower PSA progression-free survival (PFS) to D (P<0.001), and better to CZ (P=0.002). High ESRP1 expression was independently associated with longer PSA-PFS (P<0.001) and radiologic-PFS (P=0.001) in D and shorter PSA-PFS in the CZ cohort (P=0.041). High SYP expression was independently associated with lower PSA-PFS in D (P=0.003) and overall survival (OS) in CZ (P=0.002), and high EZH2 expression was associated with adverse OS in D-treated patients (P=0.013). In conclusion, EMT profile in primary tumor is differentially associated with D or CZ benefit and NE dedifferentiation correlates with adverse taxanes clinical outcome.

Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA), a deadly malignancy of the bile ducts, can be classified based on its anatomical location into either intrahepatic (iCCA) or extrahepatic (eCCA), each with different pathogenesis and clinical management. There is limited understanding of the molecular landscape of eCCA and no targeted therapy with clinical efficacy has been approved. We aimed to provide a molecular classification of eCCA and identify potential targets for molecular therapies. METHODS: An integrative genomic analysis of an international multicenter cohort of 189 eCCA cases was conducted. Genomic analysis included whole-genome expression, targeted DNA-sequencing and immunohistochemistry. Molecular findings were validated in an external set of 181 biliary tract tumors from the ICGC. RESULTS: KRAS (36.7%), TP53 (34.7%), ARID1A (14%) and SMAD4 (10.7%) were the most prevalent mutations, with ∼25% of tumors having a putative actionable genomic alteration according to OncoKB. Transcriptome-based unsupervised clustering helped us define 4 molecular classes of eCCA. Tumors classified within the Metabolic class (19%) showed a hepatocyte-like phenotype with activation of the transcription factor HNF4A and enrichment in gene signatures related to bile acid metabolism. The Proliferation class (23%), more common in patients with distal CCA, was characterized by enrichment of MYC targets, ERBB2 mutations/amplifications and activation of mTOR signaling. The Mesenchymal class (47%) was defined by signatures of epithelial-mesenchymal transition, aberrant TGFß signaling and poor overall survival. Finally, tumors in the Immune class (11%) had a higher lymphocyte infiltration, overexpression of PD-1/PD-L1 and molecular features associated with a better response to immune checkpoint inhibitors. CONCLUSION: An integrative molecular characterization identified distinct subclasses of eCCA. Genomic traits of each class provide the rationale for exploring patient stratification and novel therapeutic approaches. LAY SUMMARY: Targeted therapies have not been approved for the treatment of extrahepatic cholangiocarcinoma. We performed a multi-platform molecular characterization of this tumor in a cohort of 189 patients. These analyses revealed 4 novel transcriptome-based molecular classes of extrahepatic cholangiocarcinoma and identified ∼25% of tumors with actionable genomic alterations, which has potential prognostic and therapeutic implications.

Differentiation of Upper Urinary Tract Lesions Using MDCT: Benign Vs Malignant.

RATIONALE AND OBJECTIVES: To evaluate the accuracy of multiple detector computed tomography (MDCT) in differentiating benign and malignant lesions of upper urinary tract (UUT). MATERIALS AND METHODS: Fifty-four patients with 55 suspected UUT lesions were included in the study. All patients underwent MDCT scan with nephrographic and excretory phases. The unenhanced phase was also performed in 38 cases. The final diagnosis was made by histology in 48 lesions: 43-after surgery, 5-after biopsy and by MDCT follow-up over at least 15 months in the remaining 7 lesions. The following CT features were evaluated: number of lesions, lesion appearance (mass or wall thickening), presence of calcifications, internal border appearance (smooth or irregular), and size and enhancement (presence or absence). The relationship between imaging characteristics and pathology (benign vs malignant) was assessed with logistic regression, univariable diagnostic accuracy, and with classification and regression tree analysis. RESULTS: Patients with mass morphology had a significantly higher probability of malignancy (odds ratio [OR]: 3.73, 95%CI: 1.02-13.72, p = 0.047) compared to patients with thickened wall morphology. The presence of an irregular internal border was also significantly associated with malignancy (OR: 12.14, 95%CI: 2.95-50.06, p < 0.001). No significant associations were found between malignancy and lesion size (p = 0.29), calcifications (p = 0.93) or enhancement (p = 0.68). CONCLUSION: Mass morphology and irregular internal border are reliable signs to suggest malignancy in UUT lesions.

mRNA Detection in Anal Cytology: A Feasible Approach for Anal Cancer Screening in Men Who Have Sex with Men Living With HIV.

There is growing interest in anal cancer screening strategies. However, cytological/molecular evaluation of anal samples is challenging. We aimed to determine the feasibility of detecting, in anal liquid-based cytologies, the expression of biomarkers involved in the cell cycle disturbance elicited by human papillomavirus (HPV). The accuracy of this approach in the identification of high-grade squamous intraepithelial lesions/anal intraepithelial neoplasia grade2-3 (HSIL/AIN2-3) was also evaluated. 215 anal cytologies from men having sex with men living with human immunodeficiency virus were evaluated. Patients showing concordant cytological and anoscopy-directed biopsy diagnosis were selected: 70 with negative cytology and HPV test, 70 with low-grade SIL (LSIL/AIN1) cytology and biopsy, and 75 with cytology and biopsy of HSIL/AIN2-3. CDKN2A/p16, MKI67 and TOP2A mRNA expression was analyzed. HPV detection was performed with Xpert HPV Assay (Cepheid, Sunnyvale, CA, USA). HSIL/AIN2-3 showed higher expression for the biomarkers than LSIL/AIN1 or negative samples. The specificity for HSIL/AIN2-3 detection for a sensitivity established at 70% was 44.7% (95%confidence interval [CI] 36.5-53.2) for TOP2A and MKI67 and 54.5% (95%CI 46.0-62.8%) for CDKN2A/p16. mRNA detection of cell biomarkers in anal liquid-based cytology is feasible. Further studies are warranted to confirm if strategies based on mRNA detection have any role in anal cancer screening.

HPV-negative tumors of the uterine cervix.

Human papillomaviruses (HPV) are the causative agents of virtually all cervical carcinomas. Nevertheless, a small proportion of cervical cancer are negative for HPV, although the significance of this finding remains unclear. We aimed to provide insight into the differential clinico-pathological characteristics of this unusual subset of HPV-negative cervical cancer. We performed HPV-DNA detection using a highly sensitive PCR test (SPF10) and p16 immunostaining in 214 cervical carcinomas specimens from women treated at the Gynecological Oncology Unit of the Hospital Clinic (Barcelona, Spain) from 2012 to 2015. The clinical and pathological characteristics, including disease-free survival and overall survival, of HPV-negative and -positive cervical carcinomas were compared. Twenty-one out of 214 tumors (10%) were negative for HPV DNA. HPV-negative tumors were more frequently of the non-squamous type (9/21, 43% vs. 37/193, 19%; p < 0.01) and showed negative p16 staining (9/21; 43% vs. 7/193; 4%; p < 0.01). HPV-negative tumors were more frequently diagnosed at advanced FIGO stage (19/21, 91% vs. 110/193, 57%; p < 0.01) and more frequently had lymph node metastases (14/21, 67% vs. 69/193, 36%; p < 0.01). Patients with HPV-negative cervical cancer had a significantly worse disease-free survival (59.8 months, 95% confidence interval 32.0-87.6 vs. 132.2 months, 95% confidence interval 118.6-145.8; p < 0.01) and overall survival (77.0 months, 95% confidence interval 47.2-106.8 vs. 153.8 months, 95% confidence interval 142.0-165.6; p = 0.01) than women with HPV-positive tumors. However, only advanced FIGO stage and lymph node metastases remained associated with a poor disease-free survival and overall survival on multivariate analysis. In conclusion, our results suggest that a low percentage of cervical cancer arise via an HPV-independent pathway. These HPV-negative tumors are diagnosed at advanced stages, show higher prevalence of lymph nodes metastases and have an impaired prognosis.

Molecular predictors of prevention of recurrence in HCC with sorafenib as adjuvant treatment and prognostic factors in the phase 3 STORM trial.

OBJECTIVE: Sorafenib is the standard systemic therapy for advanced hepatocellular carcinoma (HCC). Survival benefits of resection/local ablation for early HCC are compromised by 70% 5-year recurrence rates. The phase 3 STORM trial comparing sorafenib with placebo as adjuvant treatment did not achieve its primary endpoint of improving recurrence-free survival (RFS). The biomarker companion study BIOSTORM aims to define (A) predictors of recurrence prevention with sorafenib and (B) prognostic factors with B level of evidence. DESIGN: Tumour tissue from 188 patients randomised to receive sorafenib (83) or placebo (105) in the STORM trial was collected. Analyses included gene expression profiling, targeted exome sequencing (19 known oncodrivers), immunohistochemistry (pERK, pVEGFR2, Ki67), fluorescence in situ hybridisation (VEGFA) and immunome. A gene signature capturing improved RFS in sorafenib-treated patients was generated. All 70 RFS events were recurrences, thus time to recurrence equalled RFS. Predictive and prognostic value was assessed using Cox regression models and interaction test. RESULTS: BIOSTORM recapitulates clinicopathological characteristics of STORM. None of the biomarkers tested (related to angiogenesis and proliferation) or previously proposed gene signatures, or mutations predicted sorafenib benefit or recurrence. A newly generated 146-gene signature identifying 30% of patients captured benefit to sorafenib in terms of RFS (p of interaction=0.04). These sorafenib RFS responders were significantly enriched in CD4+ T, B and cytolytic natural killer cells, and lacked activated adaptive immune components. Hepatocytic pERK (HR=2.41; p=0.012) and microvascular invasion (HR=2.09; p=0.017) were independent prognostic factors. CONCLUSION: In BIOSTORM, only hepatocytic pERK and microvascular invasion predicted poor RFS. No mutation, gene amplification or previously proposed gene signatures predicted sorafenib benefit. A newly generated multigene signature associated with improved RFS on sorafenib warrants further validation. TRIAL REGISTRATION NUMBER: NCT00692770.

IGF2 Is Up-regulated by Epigenetic Mechanisms in Hepatocellular Carcinomas and Is an Actionable Oncogene Product in Experimental Models.

BACKGROUND & AIMS: Effective treatments are urgently needed for hepatocellular carcinoma (HCC), which is usually diagnosed at advanced stages. Signaling via the insulin-like growth factor (IGF) pathway is aberrantly activated in HCC by IGF2 overexpression. We aimed to elucidate the mechanism of IGF2 overexpression and its oncogenic activities and evaluate the anti-tumor effects of reducing IGF2 signaling. METHODS: We obtained 228 HCC samples from patients who underwent liver resection, 168 paired non-tumor adjacent cirrhotic liver samples, and 10 non-tumor liver tissues from patients undergoing resection for hepatic hemangioma. We analyzed gene expression, microRNA, and DNA methylation profiles for all samples, focusing on genes in the IGF signaling pathway. IGF2 was expressed in SNU449 and PLC5 HCC cells and knocked down with small hairpin RNAs in Hep3B and Huh7 cell lines. We analyzed these cells for proliferation, apoptosis, migration, and colony formation. We performed studies in mice engineered to express Myc and Akt1 in liver, which develop liver tumors, with or without hepatic expression of Igf2. Mice with xenograft tumors grown from HCC cells were given a monoclonal antibody against IGF1 and IGF2 (xentuzumab), along with sorafenib; tumor growth was measured and tissues were analyzed by immunohistochemistry and immunoblots. RESULTS: Levels of IGF2 messenger RNA and protein were increased >20-fold in 15% of human HCC tissues compared with non-tumor liver tissues. Methylation at the fetal promoters of IGF2 was reduced in the HCC samples and cell lines that overexpressed IGF2, compared with those that did not overexpress this gene, and non-tumor tissues. Tumors that overexpressed IGF2 had gene expression patterns significantly associated with hepatic progenitor cell features, stellate cell activation, NOTCH signaling, and an aggressive phenotype (P < .0001). In mice engineered to express Myc and Akt1 in liver, co-expression of Igf2 accelerated formation of liver tumors, compared to mice with livers expressing only Myc and Akt1, and shortened survival times (P = .02). The antibody xentuzumab blocked phosphorylation of IGF1 receptor in HCC cell lines and reduced their proliferation and colony formation. In mice with xenograft tumors, injection of xentuzumab, with or without sorafenib, slowed tumor growth and increased survival times compared to vehicle or sorafenib alone. Xentuzumab inhibited phosphorylation of IGF1 receptor and AKT and reduced decreased tumor vascularization compared with vehicle. CONCLUSIONS: A large proportion of HCC samples were found to overexpress IGF2, via demethylation of its fetal promoter. Overexpression of IGF2 accelerates formation of liver tumors in mice with hepatic expression of MYC and AKT1, via activation of IGF1 receptor signaling. An antibody against IGF1 and IGF2 slows growth of xenograft tumors and increases survival of these mice.