Genome-wide Analysis Of Copy Number Alterations Research Articles

Abstract 3410: Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms

Abstract Background: Classical Hodgkin Lymphoma (cHL) is generally highly curable with standard frontline therapies, although about 20% of the patients relapse or become refractory after initial treatment. cHL hallmark is the presence of morphologically characteristic malignant Hodgkin and Reed-Sternberg (HRS) cells that represent only a small fraction (about 1%) of the surrounding non-malignant environment. Genetic alterations of HRS cells are potentially a precious source of information to develop new treatments or prognostic biomarkers. In this perspective, low tumor cellularity, worsened by DNA degradation of FFPE samples, poses technical challenges to unravel malignant cells genetic alterations. Hereby we present new insights on purified HRS single cells obtained through highly automated platforms, providing precise observation of tumor genetic alterations. Methods: FFPE tissue sections from 5 cHL patients were dissociated down to single cell suspensions. Cells were immunofluorescently labeled using anti-CD30-FITC and anti-PD-L1-PE antibodies. HRS cells, along with normal leukocytes, were selected on the basis of morphological and immunofluorescence criteria, and isolated using DEPArray™ NxT (Menarini Silicon Biosystems, MSB). Customized, high-throughput automated protocols were developed and implemented on STARLet liquid handler (Hamilton Life Sciences) to amplify isolated purified single cells genomic DNA and to generate genome-wide copy-number alterations (CNAs) profiles using Ampli1™ WGA and Ampli1™LowPass kits (MSB), respectively. Results: More than 150 HRS cells were isolated from the 5 patient samples, from which CNA profiles were obtained. HRS cells presented extensive gains and losses across the whole genome, while leukocytes displayed flat profiles as expected. HRS cells clustered coherently with patients, revealing a high degree of heterogeneity of CNA profiles among different patients. However some commonalities across the patients genomes were identified. In particular, gains and amplification were detected in PD-L1, PD-L2 and JAK2 region (9p24), as well as gains and losses in regions where REL and other genes involved in NF-kB pathway map. Conclusions: Leveraging on high throughput automated platforms and single cells isolation, the described method enabled cHL genome-wide genetic analysis at a single cell level, overcoming the intrinsic limitations of low-frequency of HRS and DNA degradation due to FFPE samples. Furthermore, unprecedented data on single HRS cells were described, opening up to a new approach to understand tumor diversity and to potentially develop personalized therapeutic strategies for cHL patients. Citation Format: Andrea Raspadori, Paola Tononi, Chiara Mangano, Marianna Garonzi, Claudio Forcato, Chiara Bolognesi, Genny Buson, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3410.

T‐cell lymphoblastic lymphoma shows differences and similarities with T‐cell acute lymphoblastic leukemia by genomic and gene expression analyses

T-cell acute lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) share common morphological and immunophenotypic features and are treated with similar therapeutic approaches. Nonetheless, they show distinct clinical presentations, suggesting that they may represent two different biological entities. To investigate the genetic characteristics of T-LBL and T-ALL, we used genomic and transcriptional profiling approaches. Genome-wide gene expression profiling, performed on 20 T-LBL and 10 T-ALL diagnostic specimens, revealed that the two malignancies shared a large fraction of their transcriptional profile while a subset of genes appeared to be differentially expressed in T-LBL versus T-ALL. This signature included genes involved in chemotactic responses and angiogenesis, which may play a role in tumor cell localization. Genome-wide copy number alteration analysis was performed on a subset of the samples analyzed by gene expression profiling and detected 41 recurrently altered genetic loci. Although most aberrations were found in both entities, several were selectively identified in T-LBL or T-ALL. In addition, NOTCH1 mutational status was found to correlate with a subset of genetic aberrations. Taken together, these results suggest that T-LBL and T-ALL are indeed two distinct diseases with unique transcriptional and genetic characteristics.

Chromosomal Instability and Copy Number Alterations in Barrett's Esophagus and Esophageal Adenocarcinoma

Chromosomal instability, as assessed by many techniques, including DNA content aneuploidy, loss of heterozygosity, and comparative genomic hybridization, has consistently been reported to be common in cancer and rare in normal tissues. Recently, a panel of chromosome instability biomarkers, including loss of heterozygosity and DNA content, has been reported to identify patients at high and low risk of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA), but required multiple platforms for implementation. Although chromosomal instability involving amplifications and deletions of chromosome regions have been observed in nearly all cancers, copy number alterations (CNA) in premalignant tissues have not been well characterized or evaluated in cohort studies as biomarkers of cancer risk. We examined CNAs in 98 patients having either BE or EA using Bacterial Artificial Chromosome (BAC) array comparative genomic hybridization to characterize CNAs at different stages of progression ranging from early BE to advanced EA. CNAs were rare in early stages (less than high-grade dysplasia) but were progressively more frequent and larger in later stages (high-grade dysplasia and EA), including high-level amplifications. The number of CNAs correlated highly with DNA content aneuploidy. Patients whose biopsies contained CNAs involving >70 Mbp were at increased risk of progression to DNA content abnormalities or EA (hazards ratio, 4.9; 95% confidence interval, 1.6-14.8; P = 0.0047), and the risk increased as more of the genome was affected. Genome-wide analysis of CNAs provides a common platform for the evaluation of chromosome instability for cancer risk assessment as well as for the identification of common regions of alteration that can be further studied for biomarker discovery.