Primary mediastinal large B-cell lymphoma is characterized by large-scale copy-neutral loss of heterozygosity.

Development of primary mediastinal B-cell lymphoma (PMBL) is driven by cumulative genomic aberrations. We discovered a unique copy-neutral loss of heterozygosity (CN-LOH) landscape of PMBL which distinguishes this tumor from other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma. Using single nucleotide polymorphism array analysis we identified large-scale CN-LOH lesions in 91% (30/33) of diagnostic PMBLs and both investigated PMBL-derived cell lines. Altogether, the cohort showed 157 extra-large (25.3-248.4 Mb) CN-LOH lesions affecting up to 14 chromosomes per case (mean of 4.4) and resulting in a reduction of heterozygosity an average of 9.9% (range 1.3-51%) of the genome. Predominant involvement of terminal chromosomal segments suggests the implication of B-cell specific crossover events in the pathogenesis of PMBL. Notably, CN-LOH stretches non-randomly clustered on 6p (60%), 15 (37.2%), and 17q (40%), and frequently co-occurred with homozygous mutations in the MHC I (6p21), B2M (15q15), and GNA13 (17q23) genes, respectively, as shown by preliminary whole-exome/genome sequencing data. Altogether, our findings implicate CN-LOH as a novel and distinct mutational process contributing to the molecular pathogenesis of PMBL. The aberration acting as "second hit" in the Knudson hypothesis, ranks as the major mechanism converting to homozygosity the PMBL-related driver genes. Screening of the cohort of 199 B cell leukemia/lymphoma whole-genomes revealed significant differences in the CN-LOH landscape of PMBL and other B-cell malignancies, including the biologically related diffuse large B-cell lymphoma.

Cancer Sample Analysis Utilizing Single-Nucleotide Polymorphism Array and Array Comparative Genomic Hybridization.

Chromosomal microarray, including single-nucleotide polymorphism (SNP) array and array comparative genomic hybridization (aCGH), enables the detection of DNA copy-number loss and/or gain associated with unbalanced chromosomal aberrations. In addition, SNP array and aCGH with SNP component also detect copy-neutral loss of heterozygosity (CN-LOH). Here we describe the chromosomal microarray procedure from the sample preparation using extracted DNA to the scanning of the array chip.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

SNP array genomic analysis of matched pairs of brain and liver metastases in primary colorectal cancer.

PURPOSE: Brain metastasis formation is a rare and late event in colorectal cancer (CRC) patients and associated with poor survival. In contrast to other metastatic sites, the knowledge on chromosomal aberrations in brain metastases is very limited. METHODS: Therefore, we carried out single nucleotide polymorphism (SNP) array analyses on matched primary CRC and brain metastases of four patients as well as on liver metastases of three patients. RESULTS: Brain metastases showed more chromosomal aberrations than primary tumors or liver metastases. Commonly occurring aberrations were gain of 8q11.1-q24.3 (primary CRC), gain of 13q12.13-q12.3 (liver metastases), and gain of 20q11.1-q13.33 (brain metastases). Furthermore, we found one copy-neutral loss of heterozygosity (cn-LOH) region on chromosome 3 in primary CRC, three cn-LOH regions in liver metastases and 23 cn-LOH regions in brain metastases, comprising 26 previously undescribed sites. CONCLUSION: The more frequent occurrence of cn-LOHs and subsequently affected genes in brain metastases shed light on the pathophysiology of brain metastasis formation. Further pairwise genetic analyses between primary tumors and their metastases will help to define the role of affected genes in cn-LOH regions.

Comparison of the copy-neutral loss of heterozygosity identified from whole-exome sequencing data using three different tools.

Loss of heterozygosity (LOH) is a genomic aberration. In some cases, LOH can be generated without changing the copy number, which is called copy-neutral LOH (CN-LOH). CN-LOH frequently occurs in various human diseases, including cancer. However, the biological and clinical implications of CN-LOH for human diseases have not been well studied. In this study, we compared the performance of CN-LOH determination using three commonly used tools. For an objective comparison, we analyzed CN-LOH profiles from single-nucleotide polymorphism array data from 10 colon adenocarcinoma patients, which were used as the reference for comparison with the CN-LOHs obtained through whole-exome sequencing (WES) data of the same patients using three different analysis tools (FACETS, Nexus, and Sequenza). The majority of the CN-LOHs identified from the WES data were consistent with the reference data. However, some of the CN-LOHs identified from the WES data were not consistent between the three tools, and the consistency with the reference CNLOH profile was also different. The Jaccard index of the CN-LOHs using FACETS (0.84 ± 0.29; mean value, 0.73) was significantly higher than that of Nexus (0.55 ± 0.29; mean value, 0.50; p = 0.02) or Sequenza (0 ± 0.41; mean value, 0.34; p = 0.04). FACETS showed the highest area under the curve value. Taken together, of the three CN-LOH analysis tools, FACETS showed the best performance in identifying CN-LOHs from The Cancer Genome Atlas colon adenocarcinoma WES data. Our results will be helpful in exploring the biological or clinical implications of CN-LOH for human diseases.

Copy Neutral LOH Affecting the Entire Chromosome 6 Is a Frequent Mechanism of HLA Class I Alterations in Cancer.

Total or partial loss of HLA class I antigens reduce the recognition of specific tumor peptides by cytotoxic T lymphocytes favoring cancer immune escape during natural tumor evolution. These alterations can be caused by genomic defects, such as loss of heterozygosity at chromosomes 6 and 15 (LOH-6 and LOH-15), where HLA class I genes are located. There is growing evidence indicating that LOH in HLA contributes to the immune selection of HLA loss variants and influences the resistance to immunotherapy. Nevertheless, the incidence and the mechanism of this chromosomal aberration involving HLA genes has not been systematically assessed in different types of tumors and often remains underestimated. Here, we used SNP arrays to investigate the incidence and patterns of LOH-6 and LOH-15 in a number of human cancer cell lines and tissues of different histological types. We observed that LOH in HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 was observed more often and was associated with homozygous HLA genotype and partial HLA loss of expression. We also discuss the immunologic and clinical implications of LOH in HLA on tumor clonal expansion and association with the cancer recurrence after treatment.

Renal angiomyolipoma (AML) harboring a missense mutation of TSC2 with copy-neutral loss of heterozygosity (CN-LOH).

Angiomyolipoma (AML) is classified as a perivascular epithelioid cell neoplasm, mostly occurring in the kidney. Twenty percent of patients with renal AML have tuberous sclerosis complex (TSC) caused by germline variation in the TSC1 or TSC2 gene. In this paper, we report the first case of renal AML harboring somatic missense mutations of the TSC2 gene and concomitant copy-neutral loss of heterozygosity (CN-LOH). The patient presented with solitary renal AML and pulmonary lymphangiomyomatosis and without other findings suggestive of TSC. Exome sequencing analysis of the renal AML, however, identified a pathogenic somatic missense mutation in the TSC2 gene (NM_000548:c.5228G>A:p. R1743Q), although no other somatic mutation was detected. Furthermore, no germline mutation in TSC1 or TSC2 was detected. Interestingly, the mutant allele ratio was too high for a somatic heterozygous mutation without loss of heterozygosity (LOH). Furthermore, no copy number variation was detected around the TSC2 locus (16p13.3). To clarify the allelic status, we analyzed heterozygous single-nucleotide polymorphisms (SNPs) in chromosome 16. In these SNPs, an unbalanced allele ratio was accumulated inside the 16p13.3 region. These results suggested copy-neutral LOH (CN-LOH). Consequently, we concluded that the missense mutation of the TSC2 gene and CN-LOH of the TSC2 locus caused renal AML.

Assessing copy number abnormalities and copy-neutral loss-of-heterozygosity across the genome as best practice in diagnostic evaluation of acute myeloid leukemia: An evidence-based review from the cancer genomics consortium (CGC) myeloid neoplasms working group.

Structural genomic abnormalities, including balanced chromosomal rearrangements, copy number gains and losses and copy-neutral loss-of-heterozygosity (CN-LOH) represent an important category of diagnostic, prognostic and therapeutic markers in acute myeloid leukemia (AML). Genome-wide evaluation for copy number abnormalities (CNAs) is at present performed by karyotype analysis which has low resolution and is unobtainable in a subset of cases. Furthermore, examination for possible CN-LOH in leukemia cells is at present not routinely performed in the clinical setting. Chromosomal microarray (CMA) analysis is a widely available assay for CNAs and CN-LOH in diagnostic laboratories, but there are currently no guidelines how to best incorporate this technology into clinical testing algorithms for neoplastic diseases including AML. The Cancer Genomics Consortium Working Group for Myeloid Neoplasms performed an extensive review of peer-reviewed publications focused on CMA analysis in AML. Here we summarize evidence regarding clinical utility of CMA analysis in AML extracted from published data, and provide recommendations for optimal utilization of CMA testing in the diagnostic workup. In addition, we provide a list of CNAs and CN-LOH regions which have documented clinical significance in diagnosis, prognosis and treatment decisions in AML.

Assessing copy number aberrations and copy-neutral loss-of-heterozygosity across the genome as best practice: An evidence-based review from the Cancer Genomics Consortium (CGC) working group for chronic lymphocytic leukemia.

The prognostic role of cytogenetic analysis is well-established in B-cell chronic lymphocytic leukemia (CLL). Approximately 80% of patients have a cytogenetic aberration. Interphase FISH panels have been the gold standard for cytogenetic evaluation, but conventional cytogenetics allows detection of additional abnormalities, including translocations, complex karyotypes and multiple clones. Whole genome copy number assessment, currently performed by chromosomal microarray analysis (CMA), is particularly relevant in CLL for the following reasons: (1) copy number alterations (CNAs) represent key events with biologic and prognostic significance; (2) DNA from fresh samples is generally available; and (3) the tumor burden tends to be relatively high in peripheral blood. CMA also identifies novel copy number variants and copy-neutral loss-of-heterozygosity (CN-LOH), and can refine deletion breakpoints. The Cancer Genomics Consortium (CGC) Working Group for CLL has performed an extensive literature review to describe the evidence-based clinical utility of CMA in CLL. We provide suggestions for the integration of CMA into clinical use and list recurrent copy number alterations, regions of CN-LOH and mutated genes to aid in interpretation.