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The Evolution of Chronic Lymphocytic Leukemia Revealed
Publish Date: 2016-12-16

December 16, 2016, Shenzhen, China – An international, multidisciplinary team of researchers led by BGI and the U.S. National Cancer Institute conducted genetic and genomic studies of biological samples collected from a patient with chronic lymphocytic leukemia(CLL) who had over 29 years of observation and treatment history. The study presents a complete representation of the evolutionary process of the cancer and provides evidences that CLL can evolve gradually during indolent phases, and rapidly generate changes at the genome and transcriptome levels following therapy. The latest study was published in Nature Communications.

 This patient was diagnosed with CLL in 1976 and died in 2001 with a span of 29 years. The first chemotherapy was administered at the 16th year, followed by several treatments over the following decade. Samples were collected at different time points from the 8th year to the 28th year. Researchers performed SNP microarray analyses, whole genome bulk sequencing, and whole genome single-cell DNA/RNA sequencing at several selected time points, using viably frozen lymphocytes.

 At the time points prior to initial therapy the researchers identified alterations on three chromosomes: deletions at 13q14 and 6q, and an amplification at 12q. The results of single-cell DNA sequencing indicated one fundamental cell clone at this early stage, which was eliminated by initial therapy.

 "The frequent changes in copy number variation (CNV) patterns over the 16 years following initial treatment indicates the cancer cells are highly adaptable." said Dr. Michael Dean from National Cancer Institute, one of the lead authors of the study. "We don't know whether the CNVs detected after therapy were caused by genome instability induced by the cancer therapy, or were selected from the pre-existing cell clones by the treatment. The results show that single cell analysis is valuable to the understanding of the effects of cancer treatment."

 The deletions of 13q14 are important prognostic markers in CLL. In early CLL, a 2 MB deletion in 13q14 was detected, which is a good prognostic marker. Between the first and second therapies, a 12 MB homozygous deletion in 13q14 appeared. This larger deletion led to the loss of an important tumor suppressor RB1, and may have contributed to the deterioration of the patient’s condition. The analyses of single cells indicated that the larger 13q14 deletion arose from cells with early focal 13q14 deletion.

 With the high-throughput single-cell RNA preparation platform (MIRACLS), the researchers successfully performed single-cell RNA-seq on hundreds of cells from different time points. The results revealed an expression pattern with high FOS, JUN and KLF4 at presentation, which emerged at the same time as the larger 13q14 deletion. After a second course of therapy, the cancer cells developed a much greater diversity of expression profiles. During a very short disease remission, the expression patterns were similar to those of early CLL cells.

 Several signaling pathways, including B cell receptor signaling, EGFR signaling, ERKs signaling and NF-kappa B signaling, showed expression changes along disease evolution.

 "Changes in CNVs represent the cell lineages across the disease evolution," said Dr. Zhikun Zhao from BGI, the first author of the study. "We observed expression changes involving cell proliferation, migration, differentiation and immune response. These signals constituted a complex network, assisting cancer cells to rapidly adjust to a new environment. The cells that survived treatment exhibited changes that were unexpected."

 "We will apply the single-cell sequencing technology on the evolution analysis of more types of cancers. In addition, the next step will also include studies about the interactions among cancer cells, immune cells and other cells in the microenvironment, as well as the dynamic changes of cancer cells under the immunotherapy." Dr. Zhao added.


Further Reading:

Evolution of multiple cell clones over a 29-year period of a CLL patient