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DNA copy number alterations (CNAs) are promising biomarkers to predict prostate cancer (PCa) outcome. However, fluorescence in situ hybridization (FISH) cannot assess complex CNA signatures because of low multiplexing capabilities. Multiplex ligation-dependent probe amplification (MLPA) can detect multiple CNAs in a single PCR assay, but PCa-specific probe mixes available commercially are lacking. Synthetic MLPA probes were designed to target 10 CNAs relevant to PCa: 5q15-21.1 (CHD1), 6q15 (MAP3K7), 8p21.2 (NKX3-1), 8q24.21 (MYC), 10q23.31 (PTEN), 12p13.1 (CDKN1B), 13q14.2 (RB1), 16p13.3 (PDPK1), 16q23.1 (GABARAPL2), and 17p13.1 (TP53), with 9 control probes. In cell lines, CNAs were detected when the cancer genome was as low as 30%. Compared with FISH in radical prostatectomy formalin-fixed, paraffin-embedded samples (n = 18: 15 cancers and 3 matched benign), the MLPA assay showed median sensitivity and specificity of 80% and 93%, respectively, across all CNAs assessed. In the validation set (n = 40: 20 tumors sampled in two areas), the respective sensitivity and specificity of MLPA compared advantageously with FISH and TaqMan droplet digital PCR (ddPCR) when assessing PTEN deletion (FISH: 85% and 100%; ddPCR: 100% and 83%) and PDPK1 gain (FISH: 100% and 92%; ddPCR: 93% and 100%). This new PCa probe mix accurately identifies CNAs by MLPA across multiple genes using low quality and quantities (50 ng) of DNA extracted from clinical formalin-fixed, paraffin-embedded samples.

Prostate cancer is a clinically heterogeneous disease and accurately risk-stratifying patients is a key clinical challenge. We hypothesized that the concurrent identification of the DNA copy number alterations 10q23.3 (PTEN) deletion and 16p13.3 (PDPK1) gain, related to the PI3K/AKT survival pathway, would improve prognostication. We assessed PTEN deletion status using fluorescence in situ hybridization (FISH) and evaluated its clinical significance in combination with the 16p13.3 gain in a set of 332 primary radical prostatectomy cases on a tissue microarray with clinical follow-up. The PTEN deletion was detected in 34% (97/287) of the evaluable tumors and was significantly associated with high Gleason grade group (P

Identifying tumors with high metastatic potential is key to improving the clinical management of prostate cancer. Recently, we characterized a chromosome 16p13.3 gain frequently observed in prostate cancer metastases and now demonstrate the prognostic value of this genomic alteration in surgically treated prostate cancer. Dual-color FISH was used to detect 16p13.3 gain on a human tissue microarray representing 304 primary radical prostatectomy (RP) cases with clinical follow-up data. The results were validated in an external dataset. The 16p13.3 gain was detected in 42% (113/267) of the specimens scorable by FISH and was significantly associated with clinicopathologic features of aggressive prostate cancer, including high preoperative PSA (P = 0.03) levels, high Gleason score (GS, P < 0.0001), advanced pathologic tumor stage (P < 0.0001), and positive surgical margins (P = 0.009). The 16p13.3 gain predicted biochemical recurrence (BCR) in the overall cohort (log-rank P = 0.0005), and in subsets of patients with PSA ≤10 or GS ≤7 (log-rank P = 0.02 and P = 0.006, respectively). Moreover, combining the 16p13.3 gain status with standard prognostic markers improved BCR risk stratification and identified a subgroup of patients with high probability of recurrence. The 16p13.3 gain status was also associated with an increased risk of developing distant metastases (log-rank P = 0.03) further substantiating its role in prostate cancer progression. Implications: This study demonstrates the prognostic significance of the 16p13.3 genomic gain in primary prostate tumors, suggesting potential utility in the clinical management of the disease by identifying patients at high risk of recurrence who may benefit from adjuvant therapies. Mol Cancer Res; 16(1); 115–23. ©2017 AACR.

International audience; Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. © 2012 Ferrata Storti Foundation.