Rocío Salgado

Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.

Oligonucleotide Array-CGH Identifies Genomic Subgroups and Prognostic Markers for Tumor Stage Mycosis Fungoides

Mycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for Research and Treatment of Cancer (EORTC) multicenter study, the genomic profile of 41 skin biopsies from tumor stage MF (MFt) was analyzed using a high-resolution oligo-array comparative genomic hybridization platform. Seventy-six percent of cases showed genomic aberrations. The most common imbalances were gains of 7q33.3q35 followed by 17q21.1, 8q24.21, 9q34qter, and 10p14 and losses of 9p21.3 followed by 9q31.2, 17p13.1, 13q14.11, 6q21.3, 10p11.22, 16q23.2, and 16q24.3. Three specific chromosomal regions, 9p21.3, 8q24.21, and 10q26qter, were defined as prognostic markers showing a significant correlation with overall survival (OS) (P=0.042, 0.017, and 0.022, respectively). Moreover, we have established two MFt genomic subgroups distinguishing a stable group (0-5 DNA aberrations) and an unstable group (>5 DNA aberrations), showing that the genomic unstable group had a shorter OS (P=0.05). We therefore conclude that specific chromosomal abnormalities, such as gains of 8q24.21 (MYC) and losses of 9p21.3 (CDKN2A, CDKN2B, and MTAP) and 10q26qter (MGMT and EBF3) may have an important role in prognosis. In addition, we describe the MFt genomic instability profile, which, to our knowledge, has not been reported earlier.

Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas

Please cite this paper as: Epidermal growth factor receptor gene numerical aberrations are frequent events in actinic keratoses and invasive cutaneous squamous cell carcinomas. Experimental Dermatology 2010; 19: 151–153.

Molecular diagnosis of dermatofibrosarcoma protuberans: a comparison between reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization methodologies.

Dermatofibrosarcoma protuberans (DFSP) is characterized by the presence of the t(17;22)(q22;q13) that leads to the fusion of the COL1A1 and PDGFB genes. This translocation can be detected by multiplex reverse transcriptase‐polymerase chain reaction (RT‐PCR) or fluorescence in situ hybridization (FISH) techniques. We have evaluated the usefulness of a dual color dual fusion FISH probe strategy for COL1A1/PDGFB detection in a series of 103 archival DFSPs and compared the obtained results with RT‐PCR analyses. FISH and RT‐PCR were carried out on paraffin embedded tissue samples. Regarding the RT‐PCR approach, all COL1A1 exons and exon 2 of PDGFB were evaluated. Sensitivity, specificity, positive and negative predictive values were assessed considering the histological diagnosis as the gold standard. We also analyzed the relationship between the genetic findings and the clinicopathological variables of the tumors. The COL1A1/PDGFB translocation was detected in 93% of DFSP. Both techniques showed a similar specificity (100%), but FISH was more sensitive than RT‐PCR (90% vs. 72%). Regarding, clinicopathological features, a higher percentage of positive cells detected by FISH was significantly associated with the fibrosarcomatous DFSP variant (P < 0.001). Interestingly, all CD34 negative DFSP (n = 5) were positive for COL1A1/PDGFB translocation by both techniques. In conclusion, the majority of DFSP harbor the COL1A1/PDGFB translocation and FISH technique should be recommended as a routine diagnostic tool, especially in cases showing unusual histopathological subtypes and/or immunohistochemical features.

MicroRNA expression profiling and DNA methylation signature for deregulated microRNA in cutaneous T-cell lymphoma

MicroRNAs usually regulate gene expression negatively, and aberrant expression has been involved in the development of several types of cancers. Microarray profiling of microRNA expression was performed to define a microRNA signature in a series of mycosis fungoides tumor stage (MFt, n=21) and CD30+ primary cutaneous anaplastic large cell lymphoma (CD30+ cALCL, n=11) samples in comparison with inflammatory dermatoses (ID, n=5). Supervised clustering confirmed a distinctive microRNA profile for cutaneous T-cell lymphoma (CTCL) with respect to ID. A 40 microRNA signature was found in MFt including upregulated onco-microRNAs (miR-146a, miR-142-3p/5p, miR-21, miR-181a/b, and miR-155) and downregulated tumor-suppressor microRNAs (miR-200ab/429 cluster, miR-10b, miR-193b, miR-141/200c, and miR-23b/27b). Regarding CD30+ cALCL, 39 differentially expressed microRNAs were identified. Particularly, overexpression of miR-155, miR-21, or miR-142-3p/5p and downregulation of the miR-141/200c clusters were observed. DNA methylation in microRNA gene promoters, as expression regulatory mechanism for deregulated microRNAs, was analyzed using Infinium 450K array and approximately one-third of the differentially expressed microRNAs showed significant DNA methylation differences. Two different microRNA methylation signatures for MFt and CD30+ cALCL were found. Correlation analysis showed an inverse relationship for microRNA promoter methylation and microRNA expression. These results reveal a subgroup-specific epigenetically regulated microRNA signatures for MFt and CD30+ cALCL patients.

Multiple genetic copy number alterations in oral squamous cell carcinoma: study of MYC, TP53, CCDN1, EGFR and ERBB2 status in primary and metastatic tumours

Background  Oncogenesis in the oral cavity is believed to result from genetic alterations that cause a stepwise transformation of the mucosa to invasive carcinoma. In oral squamous cell carcinoma (OSCC) multiple cytogenetic abnormalities have been reported, but their practical significance remains uncertain.

MYC gene numerical aberrations in actinic keratosis and cutaneous squamous cell carcinoma

Background  The genetic alterations that drive the transition from actinic keratoses (AKs) to cutaneous squamous cell carcinomas (SCCs) have not been defined precisely. Amplification and/or overexpression of the MYC proto‐oncogene have been demonstrated in several human, malignant tumours including head and neck SCCs.

Identification of t(17;22)(q22;q13) (COL1A1/PDGFB) in dermatofibrosarcoma protuberans by fluorescence in situ hybridization in paraffin-embedded tissue microarrays

Dermatofibrosarcoma protuberans is genetically characterized by the translocation t(17;22)(q22;q13) resulting in the PDGFB/COL1A1 fusion gene. Fluorescence in situ hybridization with specific probes enables a rapid detection of this gene. In this study, the presence of the translocation t(17;22)(q22;q13) by fluorescence in situ hybridization in paraffin-embedded tissue microarrays was analyzed. Two tissue microarrays including 40 cases of dermatofibrosarcoma protuberans and 20 dermatofibromas were evaluated. Fluorescence in situ hybridization analyses were performed using a dual-color dual-fusion noncommercial probe. Clinical and histopathologic features were examined, and the association with fluorescence in situ hybridization results was assessed. A total of 29 samples of dermatofibrosarcoma protuberans and 16 of dermatofibromas were successfully evaluated. Twenty-five (86%) dermatofibrosarcoma protuberans samples were positive for the translocation, which was absent in all samples of dermatofibromas. Two of the negative dermatofibrosarcoma protuberans showed unusual, hypercellular areas with marked cytologic atypia, whereas 1 case exhibited overlap features with dermatofibroma. Tumors with fibrosarcomatous areas seemed to have a higher percentage of positive cells and the number of copies of the COL1A1/PDFGB gene. In conclusion, the COL1A1/PDGFB fusion gene was present in most of the dermatofibrosarcoma protuberans tissue samples. The detection of the translocation may be an additional diagnostic tool in cases of dermatofibrosarcoma protuberans showing nonconclusive histologic features.

Primary Cutaneous CD30+ Anaplastic Large-Cell Lymphomas Show a Heterogeneous Genomic Profile: An Oligonucleotide ArrayCGH Approach

TO THE EDITOR Primary cutaneous CD30-positive lymphoproliferative disorders are the second most common group of primary cutaneous T-cell lymphomas (Willemze et al., 2005), after the mycosis fungoides/Sézary syndrome group. The clinical and histological features of primary cutaneous anaplastic large-cell lymphoma (C-ALCL) have been well characterized, but little is known about its underlying pathogenetic and genetic alterations. Previous comparative genomic hybridization (CGH) studies focusing on C-ALCL that included a limited number of samples yielded nonhomogeneous results (Böni et al., 2000; Mao et al., 2003; Prochazkova et al., 2003; Fischer et al., 2004; Zettl et al., 2004). Recently, three studies that were based on array CGH (aCGH) and included a small number of C-ALCL patients were published (Mao et al., 2003; Laharanne et al., 2010; van Kester et al., 2010). We have investigated the genomic profile of 19 C-ALCL patients using a 60-mer 44K oligonucleotide-arrayCGH platform and compared our results with those of previous aCGH studies. C-ALCL patients were selected according to the World Health Organization–European Organization for Research and Treatment for Cancer (EORTC) classification for cutaneous lymphomas (Willemze et al., 2005). This EORTC multicenter study was conducted in the departments of pathology and dermatology of six European centers in Spain and Switzerland. The local ethics committees approved the study, and written informed consent was obtained from all patients, in accordance the Declaration of Helsinki Principles. Clinical characteristics are detailed in Supplementary Table S1 online. The study was performed with 20 10 mm snap-frozen C-ALCL samples to ensure the high quality of the DNA. Hematoxylin–eosin staining of a frozen section of each sample was performed tumor cell infiltration of at least 70%. DNA was isolated using a commercial kit as described in manufacturer’s instructions (Dneasy Blood and Tissue Kit; Qiagen, Hilden, Germany). Genomewide analysis of patient samples was conducted using the Human Genome CGH 44K microarrays (G4410B and G4426B; Agilent Technologies, Palo Alto, CA), a whole-genome platform containing 44,000 probes along the entire human genome with a mean resolution of ±75 kb. Hybridization was performed according to the manufacturer’s protocols. Data analysis was conducted as previously described (Salgado et al., 2010). Fluorescence in situ hybridization with noncommercial probes of bacterial artificial chromosome DNA clones from the CHORI bacterial artificial chromosome/PAC resource (http://bacpac. chori.org) was performed to confirm chromosomal abnormalities previously detected by aCGH in cases for which a paraffin-embedded tissue biopsy was available. Chromosomal abnormalities were detected in 17 of 19 analyzed C-ALCL samples (89.5%). Losses were more frequently detected than gains (78.9 vs. 68.4%). Mao et al. (2003) and van Kester et al. (2010) found gains more frequently than losses, whereas Laharanne et al. (2010) detected losses more frequently. The highest frequencies of chromosomal aberrations were 60% (Mao et al., 2003) and 45% (Laharanne et al., 2010; van Kester et al., 2010), in contrast to 36.8% in our present study. Regarding the smallest overlapping regions of imbalance, 15 corresponded to losses and 9 to gains. The results are summarized in Figure 1 and detailed in Supplementary Table S2 online. The specific chromosomal regions and candidate genes mapped in these regions are detailed in Table 1. The most frequent abnormalities observed were deletions located on 16q, 13q, 17p13, and 20q13. Genomic losses of 13q34 (ING1) and 16q22.11 (CTCF) detected by aCGH were confirmed by fluorescence in situ hybridization in three patients. No significant correlation between the observed clinical features and the presence of chromosomal aberrations could be demonstrated. Furthermore, no data regarding the prognostic significance of the observed genetic results were obtained. In agreement with studies by van Kester et al. (2010) and Laharanne et al. (2010), two regions were lost in our study, at 13q33.3 and 16p11.2. These regions were not detected in the first aCGH study (Mao et al., 2003), probably because they may not have been among the 57 oncogenic regions of the AmpliOnc platform. Similar to the findings of van Kester et al. (2010), we observed losses at 3p26.3, 6q21, 8p22, 13q12.11, 13q13.1, 16p11.2-16q11.2, 17p13.1, and 17p13.3 (Supplementary Table S3 online). The main concordance between our results and those of van Kester et al. (2010) was a deletion at 16q11.2. However, differences were observed for a higher frequency of 16q losses in our series, including seven genomic regions located between 16q11.2 and 16q24.3. The most Abbreviations: aCGH, array comparative genomic hybridization; C-ALCL, primary cutaneous anaplastic large-cell lymphoma; CGH, comparative genomic hybridization

CKS1B amplification is a frequent event in cutaneous squamous cell carcinoma with aggressive clinical behaviour

Genetic mechanisms giving rise to the development of cutaneous squamous cell carcinoma (cSCC) are poorly understood and development of genomic high resolution techniques has led to a better knowledge of the genetic basis of several human cancers. In this study, 16 cSCC were analyzed using array comparative genomic hybridization (arrayCGH). The most common aberrations found were gains of 3q11q13, 1q21.3q25, 13q34, and 19p13, and losses of 1p36p31, 3p24p21, 10p15q22, and 13q11q21. We detected gains (3/16) and amplification (1/16) of the 1q21.1q21.3 region. A potential candidate gene in this region, CKS1B (1q21.2), was selected for validation in an independent cohort and correlations with clinicopathological features were carried out. CKS1B gene and protein status were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in a series of 53 cSCC, 22 actinic keratoses (AK), and 10 normal skin samples. cSCC presented a higher frequency of chromosome 1 polysomy than AK (70% vs. 46%, P = 0.047). Association between CKS1B protein overexpression and both polysomy and amplification was demonstrated in cSCC (P < 0.001). Regarding amplifications, 11 cSCC patients (21%) presented CKS1B gene amplification. Interestingly, 8/11 (73%) patients who showed a CKS1B amplification had presented metastatic spread (mcSCC). Differences between the presence of CKS1B amplification and the presence or absence of mcSCC were observed (mcSCC [8/14] vs. cSCC [3/39]) (P < 0.001). Several drugs targeting CKS1B have been reported and may be useful for treating patients with cSCC and CKS1B amplifications.