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Children with adrenocortical tumors (ACTs) often present with virilization due to high tumoral androgen production, with dihydrotestosterone (DHT) as most potent androgen. Recent work revealed two pathways for DHT biosynthesis, the classic and the backdoor pathway. Usage of alternate routes for DHT production has been reported in castration-resistant prostate cancer, CAH and PCOS. To assess whether the backdoor pathway may contribute to the virilization of pediatric ACTs, we investigated seven children suffering from androgen producing tumors using steroid profiling and immunohistochemical expression studies. All cases produced large amounts of androgens of the classic and/or backdoor pathway. Variable expression of steroid enzymes was observed in carcinomas and adenomas. We found no discriminative pattern. This suggests that enhanced androgen production in pediatric ACTs is the result of deregulated steroidogenesis through multiple steroid pathways. Thus future treatments of ACTs targeting androgen overproduction should consider these novel steroid production pathways.

// Christian Schafroth 1 , Jose A. Galvan 1 , Irene Centeno 1 , Viktor H. Koelzer 1,2 , Heather E. Dawson 1,2 , Lena Sokol 1 , Gregor Rieger 1,2 , Martin D. Berger 3 , Marion Hadrich 4 , Robert Rosenberg 5 , Ulrich Nitsche 6 , Beat Schnuriger 4 , Rupert Langer 1,2 , Daniel Inderbitzin 4,7 , Alessandro Lugli 1,2 and Inti Zlobec 1 1 Translational Research Unit, Institute of Pathology, Bern University Hospital, Bern, Switzerland 2 Division of Clinical Pathology, Institute of Pathology, University of Bern, Bern, Switzerland 3 Department of Medical Oncology, Bern University Hospital, Bern, Switzerland 4 Visceral Surgery and Medicine, Bern University Hospital, Bern, Switzerland 5 Department of Surgery, Kantonsspital Baselland, Liestal, Switzerland 6 Department of Surgery, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany 7 Department of Surgery, Tiefenau Hospital, Bern, Switzerland Correspondence to: Inti Zlobec, email: // Keywords : colorectal cancer, BRAF, VE1, heterogeneity, prognosis, metastasis, Pathology Section Received : September 22, 2015 Accepted : October 04, 2015 Published : October 19, 2015 Abstract Aim: VE1 is a monoclonal antibody detecting mutant BRAF V600E protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. Methods: Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers ( n = 34), melanomas ( n = 23) and thyroid cancers ( n = 8). Two prognostic cohorts were evaluated ( n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies ( n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks ( n = 100 blocks). Results: Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age ( p = 0.0175) and MLH1 deficiency ( p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender ( p = 0.0016), right-sided tumor location ( p < 0.0001), higher tumor grade ( p < 0.0001) and mismatch repair (MMR)-deficiency ( p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. Conclusion: VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

BACKGROUND In colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration. METHODS CDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation. RESULTS Sixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988-0.997)) as well as BRAF, mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2- to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2. CONCLUSION CDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.