Genomewide Copy Number Alteration Screening of Circulating Plasma DNA

Potential for the Detection of Incipient Tumors

L. Lenaerts; P. Vandenberghe; N. Brison; H. Che; M. Neofytou; M. Verheecke; L. Leemans; C. Maggen; B. Dewaele; L. Dehaspe; S. Vanderschueren; D. Dierickx; V. Vandecaveye; F. Amant; J. R. Vermeesch


Ann Oncol. 2019;30(1):85-95. 

In This Article

Supplementary Material

Detailed Clinical and Molecular Investigations in Cases 12012016–28, 25022016–07, 02102015–03, 09022017–08, 15092016–04 and 16032017–13, Diagnosed With Cancer or a Premalignant Condition Following Deviating GIPseq Profiles.

For case 12012016–28 (74-year old, male), GIPseq profiling of cfDNA showed substantial gains and losses on multiple chromosomes (Table 1A and Figure 3A). Aberrations were highly reproducible in the two independent plasma samples, taken with a time span of 3 months. Array CGH performed on leukocyte DNA of the index confirmed the presence of the aberrations seen on chromosomes 2, 5, 7, 8 and 17 in cfDNA (Figure 3B). Subsequent WB-DWI MRI showed multiple infra- and supradiaphragmatic adenopathies suggestive of a lymphoproliferative process (Figure 23). Analysis of a peripheral blood sample pointed to a leukocytosis characterized by the presence of atypical lymphoid cells. Immunophenotyping finally led to the identification of a non-Hodgkin lymphoma type chronic lymphocytic leukemia (CLL), Rai I – Binet A.

For case 25022016–07 (65-year old, male), copy number profiling of his first plasma cfDNA sample revealed an amplification of the long arm of chromosome 3. Three months later, analysis of a second sample showed a progressive evolution with large CNVs on multiple chromosomes (Supplementary Figure S1A). Shortly after taking the second plasma sample and before GIPseq results were available for interpretation, the patient had an aberrant MRI scan in the context of a back surgery which led to the biopsy diagnosis of a classical Hodgkin lymphoma, stage II. General hematological parameters were in the normal range. Due to the rarity of Hodgkin/Reed-Sternberg cells, we resorted to FISH analysis of a FFPE biopsy section to search for genomic imbalances in tumor cells. FISH results confirmed that the gain on 3q, and losses on 11q and 17q, as detected in cfDNA in the second plasma sample, were derived from tumor DNA (Table 1A and Supplementary Figure S1B).

In the third case (case 02102015–03, 69-year old, female), the GIPseq profile of the first cfDNA sample had an elevated QS, showing multiple focal gains and losses. In the second sample, taken 2, 5 months later, deletions on chromosomes 5q and 17 emerged (Table 1A and Supplementary Figure S2A). Subsequent follow-up WB-DWI MRIs were indicative for lesions in the breast and the endometrium, but further in-depth clinical investigations (by ultrasound, mammography and biopsy) did not find evidence for a malignant process. Peripheral blood analyses pointed to an anemic condition that was interpreted in the context of the women's polymyalgia rheumatica background. Six months after analysis of the second blood sample, this woman suffered from pancytopenia and was diagnosed with myelodysplastic syndrome, with excess blasts type 1 (MDS-EB1), IPSS 2. Karyotyping of bone marrow biopsy showed an unbalanced translocation between chromosome 5 and 17, partially corresponding to the genomic imbalances observed by GIPseq (Supplementary Figure S2B).

GIPseq profiling of cell-free plasma DNA of case 09022017–08 (70-year old, male) revealed chromosomal imbalances for chromosomes 6, 16 and 19, which were progressive over time (reflected in an increasing QS score for the two plasma samples taken within a time span of 4 months) (Table 1A and Supplementary Figure S3A). General hematological parameters were in the normal range. In between the GIPseq analyses of the two consecutive blood samples, parallel and independently of our investigations, this individual was diagnosed with a gastric leiomyoma and non-Hodgkin lymphoma type mucosa associated lymphoid tissue (MALT). Array CGH on peripheral blood DNA showed a normal molecular karyotype (Table 1A), but whole-genome sequencing at a shallow sequencing depth (0,1x) of biopsy tissue suggested a correlation between the gain on 6p observed in cfDNA and that in MALT tumor DNA (Supplementary Figure S3B).

For case 15092016–04 (66-year old, male), GIPseq profiling of plasma cfDNA pointed to a gain of the entire chromosome 12 (Table 1B and Supplementary Figure S4A). This was confirmed in a second, independent blood sample, taken with a time span of 2 months. Array CGH on peripheral blood cell DNA was negative. FISH confirmed the presence of a trisomy 12 in 6% of investigated peripheral blood cells (Supplementary Figure S4B). Subsequent WB-DWI MRI examinations showed infra- and supradiaphragmatic adenopathies, indicative of a lymphoma (Supplementary Figure S4C). General peripheral blood analysis pointed to lymphocytosis, and, following immunophenotyping analyses, a diagnosis of a non-Hodgkin lymphoma type small lymphocytic lymphoma (SLL), stage III, was finally made.

For the sixth case 16032017–13 (76-year old, female), cfDNA analysis pointed to an apparent deletion of 17p and gain of 17q combined with deletion of the entire chromosome 21 (Table 1A and Supplementary Figure S5A). WB-DWI MRI detected suspicious lesions in the proximal transverse colon and the left kidney. A subsequent colonoscopy did not find evidence for a malignant process in colon, whereas a dedicated CT and MRI imaging of the kidneys pointed to the presence of a very small tumoral lesion. Given the small size and possibly benign nature of the lesion a watch and wait strategy was adopted. Peripheral blood analysis pointed to the presence of lymphocytes with aberrant morphology, indicative of a lymphoproliferative process. Immunophenotyping led to the diagnosis of a high-count monoclonal B-cell lymphocytosis (MBL). FISH analyses of peripheral blood confirmed the presence of chromosome 17 and 21 aneuploidies as observed in cfDNA (Supplementary Figure S5B).