Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling.

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Risk prediction for metastasis of clear cell renal cell carcinoma using digital multiplex ligation-dependent probe amplification.

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.

BRCAness digitalMLPA profiling predicts benefit of intensified platinum-based chemotherapy in triple-negative and luminal-type breast cancer.

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.

Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.

Subclonal TP53 copy number is associated with prognosis in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous cancer of bone marrow plasma cells with variable outcome. To assess the prognostic relevance of clonal heterogeneity of TP53 copy number, we profiled tumors from 1777 newly diagnosed Myeloma XI trial patients with multiplex ligation-dependent probe amplification (MLPA). Subclonal TP53 deletions were independently associated with shorter overall survival, with a hazard ratio of 1.8 (95% confidence interval, 1.2-2.8; P = .01). Clonal, but not subclonal, TP53 deletions were associated with clinical markers of advanced disease, specifically lower platelet counts (P < .001) and increased lactate dehydrogenase (P < .001), as well as a higher frequency of features indicative of genomic instability, del(13q) (P = .002) or del(1p) (P = .006). Biallelic TP53 loss-of-function by mutation and deletion was rare (2.4%) and associated with advanced disease. We present a framework for identifying subclonal TP53 deletions by MLPA, to improve patient stratification in MM and tailor therapy, enabling management strategies.

Frequent Coamplification of Receptor Tyrosine Kinase and Downstream Signaling Genes in Japanese Primary Gastric Cancer and Conversion in Matched Lymph Node Metastasis.

OBJECTIVE: To establish the gene copy number status of receptor tyrosine kinase (RTK) and downstream signaling (DSS) genes genes in primary gastric cancer (primGC) and matched lymph node metastases (LNmet). BACKGROUND: Evidence suggests that coamplification between RTKs and DSSs and conversion between primGC and LNmet are associated with resistance to targeted therapy. METHODS: DNA from 237 Japanese primGC and 103 matched LNmet was analyzed using a newly developed multiplex ligation-dependent probe amplification (MLPA) probemix to investigate RTK (EGFR, HER2, FGFR2, and MET) and DSS (PIK3CA, KRAS, MYC, and CCNE1) gene copy number status. Results were compared between primGC and LNmet and related to clinicopathological data including survival. RESULTS: A total of 150 (63%) primGC had either RTK or DSS amplification. DSS coamplification was more frequent than RTK coamplification in primGC and LNmets. Moreover, 70 (30%) GC showed a disconcordant RTK and/or DSS gene copy number status between primGC and LNmet, most common was negative conversion for DSS genes (n=40 GC). The presence of RTK amplification in primGC was related to poorer survival in univariate analysis (P=0.04). CONCLUSIONS: This is the first and most comprehensive study in gastric cancer investigating the concordance between gene copy number status of targetable RTKs and downstream signaling oncogenes in primGC and LNmets. Future studies need to establish whether the relative high frequency of RTK and DSS coamplification and/or the relative high rate of negative conversion in LNmet can potentially explain recent failures of RTK targeted therapy in gastric cancer patients.

Role of columnar cell lesions in breast carcinogenesis: analysis of chromosome 16 copy number changes by multiplex ligation-dependent probe amplification.

Columnar cell lesions have been proposed as precursor lesions of low-grade breast cancer. The molecular characteristic of low-grade breast neoplasia is whole-arm loss of chromosome 16q. Copy number changes of 6 genes on 16p and 20 genes on 16q were analysed by multiplex ligation-dependent probe amplification in 165 lesions of 103 patients. Twenty-three columnar cell lesions and 19 atypical ducal hyperplasia lesions arising in columnar cell lesions were included, as well as cases of usual ductal hyperplasia, blunt duct adenosis, ductal carcinoma in situ, lobular neoplasia and invasive carcinoma. Usual ductal hyperplasia and blunt duct adenosis lacked whole-arm losses of 16q. In contrast, columnar cell lesions without atypia, columnar cell lesions with atypia, atypical ductal hyperplasia, low-grade ductal carcinoma in situ and low-grade invasive carcinomas increasingly harboured whole-arm losses of 16q (17%, 27%, 47% and 57%, respectively). However, no recurrent losses in specific genes could be identified. In several patients, columnar cell lesions and atypical ductal hyperplasia harboured similar losses as related ductal carcinoma in situ or invasive carcinomas within the same breast. There were indications for 16q breakpoints near the centromere. Whole-arm gains on 16p were relatively scarce and there was no relation between whole-arm gains of 16p and progression of lesions of the low-grade breast neoplasia family. In conclusion, columnar cell lesions (with and without atypia) often harbour whole-arm losses of 16q, which underlines their role as precursors in low-grade breast carcinogenesis, in contrast with usual ductal hyperplasia and blunt duct adenosis. However, no recurrent losses in specific genes could be identified, pointing to minor events in multiple tumour suppressor genes rather than major events in a single 16q gene contributing to low-grade breast carcinogenesis.

A molecular diagnostic approach able to detect the recurrent genetic prognostic factors typical of presenting myeloma.

Risk stratification in myeloma requires an accurate assessment of the presence of a range of molecular abnormalities including the differing IGH translocations and the recurrent copy number abnormalities that can impact clinical behavior. Currently, interphase fluorescence in situ hybridization is used to detect these abnormalities. High failure rates, slow turnaround, cost, and labor intensiveness make it difficult and expensive to use in routine clinical practice. Multiplex ligation-dependent probe amplification (MLPA), a molecular approach based on a multiplex polymerase chain reaction method, offers an alternative for the assessment of copy number changes present in the myeloma genome. Here, we provide evidence showing that MLPA is a powerful tool for the efficient detection of copy number abnormalities and when combined with expression assays, MLPA can detect all of the prognostically relevant molecular events which characterize presenting myeloma. This approach opens the way for a molecular diagnostic strategy that is efficient, high throughput, and cost effective.

Renal cell carcinoma with smooth muscle stroma lacks chromosome 3p and VHL alterations.

Renal cell carcinoma with prominent smooth muscle stroma is a rare neoplasm composed of an admixture of epithelial cell with clear cytoplasm arranged in small nest and tubular structures and a stroma composed of smooth muscle. In the epithelial component, loss of chromosome 3p detected by fluorescence in situ hybridization (FISH) has been reported and on this basis these neoplasms have been viewed as variants of clear cell renal cell carcinoma. To test the validity of this classification, we have evaluated the chromosome 3 and VHL status of three of these tumors using FISH, array comparative genomic hybridization, gene sequencing, and methylation-specific multiplex ligation-dependent probe amplification analysis. None of the tumors showed deletion of chromosome 3p, VHL mutation, a significant VHL methylation, or changes in VHL copy number and all three tumors demonstrated a flat profile in the comparative genomic hybridization analysis. We conclude that renal cell carcinoma with smooth muscle stroma should be considered as an entity distinct from clear cell renal cell carcinoma.

Genetic imbalances detected by multiplex ligation-dependent probe amplification in a cohort of patients with oral squamous cell carcinoma-the first step towards clinical personalized medicine.

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.

Targeted resequencing of 9p in acute lymphoblastic leukemia yields concordant results with array CGH and reveals novel genomic alterations.

Genetic alterations of the short arm of chromosome 9 are frequent in acute lymphoblastic leukemia. We performed targeted sequencing of 9p region in 35 adolescent and adult acute lymphoblastic leukemia patients and sought to investigate the sensitivity of detecting copy number alterations in comparison with array comparative genomic hybridization (aCGH), and besides, to detect novel genetic anomalies. We found a high concordance of copy number variations (CNVs) as detected by next generation sequencing (NGS) and aCGH. By both methodologies, the recurrent deletion at CDKN2A/B locus was identified, whereas NGS revealed additional, small regions of CNVs, seen more frequently in adult patients, while aCGH was better at detecting larger CNVs. Also, by NGS, we detected novel structural variations, novel SNVs and small insertion/deletion variants. Our results show that NGS, in addition to detecting mutations and other genetic aberrations, can be used to study CNVs.

Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization are complementary techniques to detect cytogenetic abnormalities in multiple myeloma.

Multiple myeloma (MM) is a genetically heterogeneous disease with diverse clinical outcomes. Interphase fluorescence in situ hybridization (i-FISH) is the most commonly used approach to detect recurrent cytogenetic abnormalities in this malignancy. We aimed to assess the performance of multiplex ligation-dependent probe amplification (MLPA) to reveal copy number abnormalities (CNAs) in MM. Diagnostic bone marrow samples from 81 patients were analyzed using 42 MLPA probes for the following regions: 1p32-31, 1p21, 1q21.3, 1q23.3, 5q31.3, 12p13.31, 13q14, 16q12, 16q23, and 17p13. All samples were also screened by i-FISH for the presence of hyperdiploidy, deletion/monosomy of chromosome 13, deletion of TP53, disruption of the immunoglobulin heavy-chain gene, t(4;14), t(11;14), t(14;16), t(8;14), gain of 5q and abnormalities of chromosome 1. A total of 245 alterations were detected in 79 cases (98%). Investigating the same aberrations, the two methods showed a congruency of higher than 90%. A low proportion of cells with the relevant abnormality, focal CNAs and unmatched probes were responsible for the discrepancies. MLPA revealed 95 CNAs not detected by i-FISH providing additional information in 53 cases (65%). Scrutiny of CNAs on chromosome 1, using more than 20 probes, revealed significant heterogeneity in size and location, and variable intra-chromosomal and intra-clonal rates of loss or gain. Our results suggest that MLPA is a reliable high-throughput technique to detect CNAs in MM. Since balanced aberrations are key to prognostic classification of this disease, MLPA and i-FISH should be applied as complementary techniques in diagnostic pathology.

Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness.

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.

The genomic landscape of teenage and young adult T-cell acute lymphoblastic leukemia.

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.

Copy number evolution and its relationship with patient outcome-an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial.

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis-relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

Genomic profiling of chondrosarcoma: chromosomal patterns in central and peripheral tumors.

PURPOSE: Histologic grade is currently the best predictor of clinical course in chondrosarcoma patients. Grading suffers, however, from extensive interobserver variability and new objective markers are needed. Hence, we have investigated DNA copy numbers in chondrosarcomas with the purpose of identifying markers useful for prognosis and subclassification. EXPERIMENTAL DESIGN: The overall pattern of genomic imbalances was assessed in a series of 67 chondrosarcomas using array comparative genomic hybridization. Statistical analyses were applied to evaluate the significance of alterations detected in subgroups based on clinical data, morphology, grade, tumor size, and karyotypic features. Also, the global gene expression profiles were obtained in a subset of the tumors. RESULTS: Genomic imbalances, in most tumors affecting large regions of the genome, were found in 90% of the cases. Several apparently distinctive aberrations affecting conventional central and peripheral tumors, respectively, were identified. Although rare, recurrent amplifications were found at 8q24.21-q24.22 and 11q22.1-q22.3, and homozygous deletions of loci previously implicated in chondrosarcoma development affected the CDKN2A, EXT1, and EXT2 genes. The chromosomal imbalances in two distinct groups of predominantly near-haploid and near-triploid tumors, respectively, support the notion that polyploidization of an initially hyperhaploid/hypodiploid cell population is a common mechanism of chondrosarcoma progression. Increasing patient age as well as tumor grade were associated with adverse outcome, but no copy number imbalance affected metastasis development or tumor-associated death. CONCLUSION: Despite similarities in the overall genomic patterns, the present findings suggest that some regions are specifically altered in conventional central and peripheral tumors, respectively.

High-Throughput Molecular Cancer Cell Line Characterization Using Digital Multiplex Ligation-Dependent Probe Amplification for Improved Standardization of in Vitro Research.

Tumor cell lines are widely used for cancer research, but challenges regarding quality control of cell line identity, cross contamination, and tumor somatic molecular stability remain, demanding novel approaches beyond conventional short tandem repeat profiling. A total of 21 commonly used multiple myeloma cell lines obtained from public repositories were analyzed by digital multiplex ligation-dependent probe amplification (digitalMLPA) to characterize germline single-nucleotide polymorphisms, insertions/deletions, and somatic copy number aberrations (CNAs). Using generated profiles and an in-house developed analytical pipeline, blinded experiments were performed to determine capability of digitalMLPA to predict cell line identity and potential spike-in DNA contamination in 41 anonymized cell line samples. The dominant cell line was correctly identified in all cases, and cross contamination was correctly detected in 33 of 37 samples with spike-in DNA; there were no false-positive predictions. The four samples in which spike in was not detected all carried low levels of contamination (1%), whereas levels of contamination ≥5% were correctly identified in all cases. Unsupervised clustering of CNA profiles identified shared commonalities that correlated with initiating Ig heavy locus translocation events. Longitudinal CNA assessment of nine cell lines revealed changes under standard culturing conditions not detected by insertion/deletion profiling alone. Results suggest that digitalMLPA can be utilized as a high-throughput tool for advanced quality assurance for in vitro cancer research.

Working together for the family: determination of HER oncogene co-amplifications in breast cancer.

HER2 is a well-studied tyrosine kinase (TK) membrane receptor which functions as a therapeutic target in invasive ductal breast carcinomas (IDC). The standard of care for the treatment of HER2-positive breast is the antibody trastuzumab. Despite specific treatment unfortunately, 20% of primary and 70% of metastatic HER2 tumors develop resistance. HER2 belongs to a gene family, with four members (HER1-4) and these members could be involved in resistance to anti-HER2 therapies. In this study we designed a probemix to detect the amplification of the four HER oncogenes in a single reaction. In addition, we developed a protocol based on the combination of MLPA with ddPCR to detect the tumor proportion of co-amplified HERs. On 111 IDC, the HER2 MLPA results were validated by FISH (Adjusted r 2 = 0,91, p < 0,0001), CISH (Adjusted r 2 = 0,938, p < 0,0001) and IHC (Adjusted r 2 = 0,31, p < 0,0001). HER1-4 MLPA results were validated by RT-qPCR assays (Spearman Rank test p < 0,05). Of the 111 samples, 26% presented at least one HER amplified, of which 23% showed co-amplifications with other HERs. The percentage of cells with HER2 co-amplified varied among the tumors (from 2-72,6%). Independent in-silico findings show that the outcome of HER2+ patients is conditioned by the status of HER3 and HER4. Our results encourage further studies to investigate the relationship with patient's response to single or combined treatment. The approach could serve as proof of principle for other tumors in which the HER oncogenes are involved.

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors.

BACKGROUND: Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas. METHODS: Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5-192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest. RESULTS: Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting gene, HDGF, confirmed that its expression was higher than in control samples. However, no association between HDGF expression and patient survival was observed. CONCLUSION: We conclude that array CGH and integration analysis proved to be effective methods to identify chromosome regions and novel target genes involved in the tumorigenesis of ESFT.