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Cancer is caused by an accumulation of somatic mutations and copy number alterations (CNAs). Besides mutations, these copy number changes are key characteristics of cancer development. Nonetheless, some tumors show hardly any CNAs, a remarkable phenomenon in oncogenesis. Head and neck squamous cell carcinomas (HNSCCs) arise by either exposure to carcinogens, or infection with the human papillomavirus (HPV). HPV-negative HNSCCs are generally characterized by many CNAs and frequent mutations in CDKN2A, TP53, FAT1, and NOTCH1. Here, we present the hallmarks of the distinct subgroup of HPV-negative HNSCC with no or few CNAs (CNA-quiet) by genetic profiling of 802 oral cavity squamous cell carcinomas (OCSCCs). In total, 73 OCSCC (9.1%) are classified as CNA-quiet and 729 as CNA-other. The CNA-quiet group is characterized by wild-type TP53, frequent CASP8 and HRAS mutations, and a less immunosuppressed tumor immune microenvironment with lower density of regulatory T cells. Patients with CNA-quiet OCSCC are older, more often women, less frequently current smokers, and have a better 5-year overall survival compared to CNA-other OCSCC. This study demonstrates that CNA-quiet OCSCC should be considered as a distinct, clinically relevant subclass. Given the clinical characteristics, the patient group with these tumors will rapidly increase in the aging population. HPV-negative head and neck squamous cell carcinomas (HNSCCs) are generally characterized by many copy number alterations (CNAs) and mutations. Here, the authors characterize a subgroup of HPV-negative HNSCC with no or few CNAs (CNA-quiet) by genomic profiling of 802 oral cavity squamous cell carcinomas. [ABSTRACT FROM AUTHOR]

Precise quantification of copy‐number alterations (CNAs) in a tumor genome is difficult. We have applied a comprehensive copy‐number analysis method, digital multiplex ligation‐dependent probe amplification (digitalMLPA), for targeted gene copy‐number analysis in clear cell renal cell carcinoma (ccRCC). Copy‐number status of all chromosomal arms and 11 genes was determined in 60 ccRCC samples. Chromosome 3p loss and 5q gain, known as early changes in ccRCC development, as well as losses at 9p and 14q were detected in 56/60 (93.3%), 31/60 (51.7%), 11/60 (18.3%), and 33/60 (55%), respectively. Through gene expression analysis, a significant positive correlation was detected in terms of 14q loss determined using digitalMLPA and downregulation of mRNA expression ratios with HIF1A and L2HGDH (P =.0253 and.0117, respectively). Patients with early metastasis (<1 y) (n = 18) showed CNAs in 6 arms (in median), whereas metastasis‐free patients (n = 34) showed those in significantly less arms (3 arms in median) (P =.0289). In particular, biallelic deletion of CDKN2A/2B was associated with multiple CNAs (≥7 arms) in 3 tumors. Together with sequence‐level mutations in genes VHL, PBRM1, SETD2, and BAP1, we performed multiple correspondence analysis, which identified the association of 9p loss and 4q loss with early metastasis (both P <.05). This analysis indicated the association of 4p loss and 1p loss with poor survival (both, P <.05). These findings suggest that CNAs have essential roles in aggressiveness of ccRCC. We showed that our approach of measuring CNA through digitalMLPA will facilitate the selection of patients who may develop metastasis. [ABSTRACT FROM AUTHOR]

Background: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T‐cell acute lymphoblastic leukemia (T‐ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T‐ALL has a specific biological‐molecular profile distinct from pediatric or adult T‐ALL. Methods: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15–26 years with primary or relapsed T‐ALL, using a combination of Genome‐Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real‐time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1–14 years), 439 TYA (15–24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search. Results: Genomic characterization of this large cohort of TYA T‐ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation – relapsed T‐ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways. Conclusions: All genetic aberrations in TYA T‐ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T‐ALL exhibits a transitional genomic profile. Analysis of matched presentation – relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub‐clones associated with drug resistance (NT5C2 and TP53 mutations) and re‐iterative mutation of known key T‐ALL drivers, including NOTCH1. [ABSTRACT FROM AUTHOR]

Background: We previously showed that BRCA-like profiles can be used to preselect individuals with the highest risk of carrying BRCA mutations but could also indicate which patients would benefit from double-strand break inducing chemotherapy. A simple, robust, and reliable assay for clinical use that utilizes limited amounts of formalin-fixed, paraffin-embedded tumor tissue to assess BRCAness status in both ER-positive and ER-negative breast cancer (BC) is currently lacking.Methods: A digital multiplex ligation-dependent probe amplification (digitalMLPA) assay was designed to detect copy number alterations required for the classification of BRCA1-like and BRCA2-like BC. The BRCA1-like classifier was trained on 71 tumors, enriched for triple-negative BC; the BRCA2-like classifier was trained on 55 tumors, enriched for luminal-type BC. A shrunken centroid-based classifier was developed and applied on an independent validation cohort. A total of 114 cases of a randomized controlled trial were analyzed, and the association of the classifier result with intensified platinum-based chemotherapy response was assessed.Results: The digitalMLPA BRCA1-like classifier correctly classified 91% of the BRCA1-like samples and 82% of the BRCA2-like samples. Patients with a BRCA-like tumor derived significant benefit of high-dose chemotherapy (adjusted hazard ratio (HR) 0.12, 95% CI 0.04-0.44) which was not observed in non-BRCA-like patients (HR 0.9, 95% CI 0.37-2.18) (p = 0.01). Analysis stratified for ER status showed borderline significance.Conclusions: The digitalMLPA is a reliable method to detect a BRCA1- and BRCA2-like pattern on clinical samples and predicts platinum-based chemotherapy benefit in both triple-negative and luminal-type BC. [ABSTRACT FROM AUTHOR]

Recurrent clonal genetic alterations are the hallmark of Acute Lymphoblastic Leukemia (ALL) and govern the risk stratification, response to treatment and clinical outcome. In this retrospective study conducted on ALL patient samples, the purpose was to estimate the copy number alterations (CNAs) in ALL by digitalMLPA (dMLPA), validation of the dMLPA data by conventional MLPA and RT-PCR, and correlation of CNAs with Minimal Residual Disease (MRD) status. The ALL patient samples (n = 151; B-ALL, n = 124 cases and T-ALL, n = 27 cases) were assessed for CNAs by dMLPA for detection of sub-microscopic CNAs and ploidy status. This assay allowed detection of ploidy changes and CNAs by multiplexing of karyotyping probes and probes covering 54 key gene targets implicated in ALL. Using the dMLPA assay, CNAs were detected in ~89% (n = 131) of the cases with 66% of the cases harboring ≥3 CNAs. Deletions in CDKN2A/B, IKZF1 , and PAX5 genes were detectable in a quarter of these cases. Heterozygous and homozygous gene deletions, and duplications were observed in genes involved in cell cycle control, tumor suppression, lineage differentiation, lymphoid signaling, and transcriptional regulators with implications in treatment response and survival outcome. Distinct CNAs profiles were evident in B-ALL and T-ALL cases. Additionally, the dMLPA assay could reliably identify ploidy status and copy number-based gene fusions (SIL-TAL1, NUP214-ABL, EBF1-PDGFRB). Cases of B-ALL with no detectable recurrent genetic abnormalities could potentially be risk stratified based on the CNA profile. In addition to the commonly used gene deletions for risk assessment (IKZF1, EBF1, CDKN2A/B), we identified a broader spectrum of gene alterations (gains of- RUNX1, LEF1, NR3C2, PAR1, PHF6; deletions of- NF1, SUZ12, MTAP) that significantly correlated with the status of MRD clearance. The CNAs detected by dMLPA were validated by conventional MLPA and showed high concordance (r = 0.99). Our results demonstrated dMLPA to be a robust and reliable alternative for rapid detection of key CNAs in newly diagnosed ALL patients. Integration of ploidy status and CNAs detected by dMLPA with cytogenetic and clinical risk factors holds great potential in further refinement of patient risk stratification and response to treatment in ALL. [ABSTRACT FROM AUTHOR]

Objectives: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods.Methods: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared.Results: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance.Conclusions: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen. [ABSTRACT FROM AUTHOR]

Current conventional treatment modalities in head and neck squamous cell carcinoma ( HNSCC) are nonselective and have shown to cause serious side effects. Unraveling the molecular profiles of head and neck cancer may enable promising clinical applications that pave the road for personalized cancer treatment. We examined copy number status in 36 common oncogenes and tumor suppressor genes in a cohort of 191 oropharyngeal squamous cell carcinomas ( OPSCC) and 164 oral cavity squamous cell carcinomas ( OSCC) using multiplex ligation probe amplification. Copy number status was correlated with human papillomavirus ( HPV) status in OPSCC, with occult lymph node status in OSCC and with patient survival. The 11q13 region showed gain or amplifications in 59% of HPV-negative OPSCC, whereas this amplification was almost absent in HPV-positive OPSCC. Additionally, in clinically lymph node-negative OSCC (Stage I-II), gain of the 11q13 region was significantly correlated with occult lymph node metastases with a negative predictive value of 81%. Multivariate survival analysis revealed a significantly decreased disease-free survival in both HPV-negative and HPV-positive OPSCC with a gain of Wnt-induced secreted protein-1. Gain of CCND1 showed to be an independent predictor for worse survival in OSCC. These results show that copy number aberrations, mainly of the 11q13 region, may be important predictors and prognosticators which allow for stratifying patients for personalized treatment of HNSCC. [ABSTRACT FROM AUTHOR]

Sequestration of the tumor suppressor retinoblastoma protein ( RB) by the Merkel cell polyomavirus ( MCV) is a crucial step in the pathogenesis of Merkel cell carcinoma ( MCC). RB expression is frequently lost, particularly in MCV-negative MCC tumors, through yet unknown mechanisms. We compared the genomic copy number changes of 13 MCV-positive and 13 -negative MCC tumors by array comparative genomic hybridization. The analysis revealed increased genomic instability, amplification of 1p34.3-1p34.2, and losses of 11p in the absence of MCV infection. Deletions of the RB1 locus were also detected at high rates in MCV-negative tumors. None of the tumors with heterozygous RB1 losses expressed RB in immunohistochemistry. RB1 promoter hypermethylation was studied with a methylation-specific multiplex ligation-dependent probe amplification technique. The RB1 promoter was methylated in all tumor specimens at CpG islands located close to the ATG start codon, albeit at low levels. The pattern of hypermethylation was similar in all MCC samples, despite RB expression, survival or MCV status. In conclusion, the frequent heterozygous losses of the RB1 locus could partly explain the decreased RB expression in MCV-negative MCC tumors, although the effects of RB1 mutations, coinciding promoter hypermethylation and, for example, mi RNA regulation, cannot be excluded. [ABSTRACT FROM AUTHOR]

We studied a series of spindle cell lipomas arising in atypical sites and showing unusual morphologic features (which we called atypical spindle cell lipoma) to assess if these lesions have the same chromosomal alterations as classical spindle cell lipoma but different from those found in atypical lipomatous tumor/well-differentiated liposarcoma. We investigated alterations of different genes in the 13q14 region and the amplification status of the MDM2 and CDK4 genes at 12q14-15 by multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analysis. In the atypical spindle cell lipomas, MLPA revealed deletions in the two nearest flanking genes of RB1 ( ITM2B and RCBTB2) and in multiple important exons of RB1. In contrast, in classical spindle cell lipomas, a less complex loss of RB1 exons was found but no deletion of ITM2B and RCBTB2. Moreover, MLPA identified a deletion of the DLEU1 gene, a finding which has not been reported earlier. We propose an immunohistochemical panel for lipomatous tumors which comprises of MDM2, CDK4, p16, Rb, which we have found useful in discriminating between atypical or classical spindle cell lipomas and other adipocytic neoplasms, especially atypical lipomatous tumor/well-differentiated liposarcoma. Our findings strengthen the link between atypical spindle cell lipoma and classical spindle cell lipoma, and differentiate them from atypical lipomatous tumor/well-differentiated liposarcoma. [ABSTRACT FROM AUTHOR]

Objective: The presence of numerical and/or structural chromosomal abnormalities is a frequent finding in clonal hematopoietic malignant disease, typically diagnosed through routine karyotyping and/or fluorescent in situ hybridization (FISH) analysis. Recently, the application of array comparative genomic hybridization (aCGH) has uncovered many new cryptic genomic copy number imbalances, most of which are now recognized as clinically useful markers of haematological malignancies. In view of the limitations of both FISH and aCGH techniques, in terms of their routine application as a first line screening test, we designed a new multiple ligation-dependent probe amplification (MLPA) probemix for use in addition to classic karyotype analysis. Methods: A novel MLPA probemix was developed to interrogate copy number changes involving chromosomal regions: 2p23-24 (MYCN, ALK), 5q32-34 (MIR145A, EBF1, MIR146A), 6q21-27, 7p12.2 (IKZF1), 7q21-36, 8q24.21 (MYC), 9p24 (JAK2 V617F point mutation), 9p21.3 (CDKN2A/2B), 9p13.2 (PAX5), 10q23 (PTEN), 11q22.3 (ATM), 12p13.2 (ETV6), 13q14 (RB1, MIR15A, DLEU2, DLEU1), 17p13.1 (TP53), and 21q22.1 (RUNX1/AML1) and was applied to DNA extracted from 313 consecutive bone marrow patient samples, referred for routine karyotype analysis. Results: More than half of the samples originated from newly investigated patients. We discovered clinically relevant genomic aberrations, involving a total of 24 patients (8%) all with a normal karyotype, which would have remained undiagnosed. Discussion: Our data clearly indicate that routine application of this MLPA screening panel, as an adjunct to karyotype analysis, provides a sensitive, robust, rapid and low-cost approach for uncovering clinically important genomic abnormalities, which would have otherwise remained undetected. [ABSTRACT FROM AUTHOR]

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material- RARB, FHIT, CSMD1, GATA4, and MTUS1-and others due to gains- MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients' outcomes, and also to guide the development of novel molecular therapies. [ABSTRACT FROM AUTHOR]

Corticosteroids can influence brain function, and glucocorticoid hormone receptors (GRs) are present in brain tissue. We observed that GR and also mineralocorticoid receptor (MR) are expressed by embryonic rat neural stem cells (NSCs). NSCs in developing ventricular epithelium were positive for GR. Stimulation of cultured NSCs with the specific receptor ligands dexamethasone and corticosterone reduced cell proliferation, shown by 5'-bromo-2-deoxy-uridine labeling. The effect ofthe hormones was dose dependent and inhibited by the GR blocker mifepristone but not by spironolactone, blocking MR. Dexamethasone inhibited the cell cycle by decreasing the levels of cyclin D1 in NSCs. The hormone-induced decline was inhibited by MG132 (benzyloxycarbonyl-leucyl-leucyl-leucinal), showing an involvement of the ubiquitin proteasome system, In keeping with this, dexamethasone increased the ubiquitination of cyclin D1. In embryonic brain, dexamethasoneinhibited cell proliferation of NSCs. This demonstratesthat embryonic NSCs are criticallyinfluenced by glucocorticoids, which can have long-term effects in the brain. [ABSTRACT FROM AUTHOR]

Introduction: Our group has previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. We have shown that the so-called BRCA1-like(aCGH) profile is also present in about half of all triple-negative sporadic breast cancers and is predictive for benefit from intensified alkylating chemotherapy. As aCGH is a rather complex method, we translated the BRCA1(aCGH) profile to a Multiplex Ligation-dependent Probe Amplification (MLPA) assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a BRCA1-like(aCGH) profile.Methods: The most important genomic regions of the original aCGH based classifier (3q22-27, 5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) were mapped to a set of 34 MLPA probes. The training set consisted of 39 BRCA1-like(aCGH) breast cancers and 45 non-BRCA1-like(aCGH) breast cancers, which had previously been analyzed by aCGH. The BRCA1-like(aCGH) group consisted of germline BRCA1-mutated cases and sporadic tumours with low BRCA1 gene expression and/or BRCA1 promoter methylation. We trained a shrunken centroids classifier on the training set and validation was performed on an independent test set of 40 BRCA1-like(aCGH) breast cancers and 32 non-BRCA1-like(aCGH) breast cancer tumours. In addition, we validated the set prospectively on 69 new triple-negative tumours.Results: BRCAness in the training set of 84 tumours could accurately be predicted by prediction analysis of microarrays (PAM) (accuracy 94%). Application of this classifier on the independent validation set correctly predicted BRCA-like status of 62 out of 72 breast tumours (86%). Sensitivity and specificity were 85% and 87%, respectively. When the MLPA-test was subsequently applied to 46 breast tumour samples from a randomized clinical trial, the same survival benefit for BRCA1-like tumours associated with intensified alkylating chemotherapy was shown as was previously reported using the aCGH assay.Conclusions: Since the MLPA assay can identify BRCA1-deficient breast cancer patients, this method could be applied both for clinical genetic testing and as a predictor of treatment benefit. BRCA1-like tumours are highly sensitive to chemotherapy with DNA damaging agents, and most likely to poly ADP ribose polymerase (PARP)-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, and works well with DNA derived from paraffin-embedded tissues. [ABSTRACT FROM AUTHOR]