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Single-Cell Sequencing Reveals Origin of Disseminated Breast Cancer Cells

NEW YORK (GenomeWeb) – Using single-cell sequencing, an international team of researchers has characterized cells isolated from breast cancer patients' bone marrow that they suspected of being micro-metastases.

The team, led by Oslo University Hospital's Bjørn Naume in Norway, sequenced the genomes of 63 single cells from the bone marrow of six breast cancer patients. By comparing copy number aberrations within these cells to cells from the patients' primary tumors, the team found that a small portion of them were disseminated tumor cells, as they reported in Genome Biology today. In addition, through reconstructing those cells' phylogeny, the researchers were able to get a glimpse into how these cells arise.

Disseminated tumor cells, co-first author Jonas Demeulemeester from the Francis Crick Institute in London said in a statement, "can lay undetected and dormant for many years, often resisting therapy, before re-activating and giving rise to a new tumor, a metastasis."

Based on immunocytochemical staining, Naume, Demeulemeester, and their colleagues isolated single cells from seven bone marrow aspirates of six breast cancer patients. Six of those aspirates were collected at diagnosis, while one was obtained three years after diagnosis. At the same time, the researchers collected seven control cells from the aspirates. From one patient, the researchers also collected a sample from a synchronous axillary lymph node metastasis. The cells were divided, based on their morphological characteristics, into tumor cell, probable hematopoietic cell, hematopoietic cell, and uncertain cell groups.

The genomes of all 63 cells were amplified and sequenced to 1.7X depth of coverage. Eleven of these cells had copy number aberration patterns similar to those of the patients' primary tumor or the lymph node metastasis, suggesting they were genuinely disseminated tumor cells. Most of those 11 cells, the researchers noted, had also been deemed to be tumor cells based on their morphology. Such DTCs came from three patients, all of whom later developed distant metastases.

"Only a subset of those cells previously believed to be cancer cells have really spread from the patient's primary tumor," Demeulemeester said. "A refined indicator means a more accurate prognosis and a more tailored therapy for patients, avoiding over- or under-treatment."

But how these cells disseminate has been unclear, the researchers said, noting that there are two theories describing how this might occur: one says DTCs leave their site of origin early and evolve independently, while the other says they detach from a major or minor subclone later on and thus have genomes that are largely similar to the primary tumor.

To reconstruct the disseminated tumor cells' likely path, the researchers sequenced the exomes of the patients' primary tumors and matched normal blood cells as well as the exome of one patient's lymph node metastasis to a 35X average depth of coverage. Overall, they uncovered 239 somatic substitutions in the seven cancer exomes, 103 of which were non-synonymous.

With these various mutations as a guide, the researchers reconstructed the tumor phylogeny for three patients.

For one patient, they found that the bulk primary tumor harbored clonal 1q and 17q DNA gains alongside a loss of chromosome 4 and a number of segmental deletions. A subclone from that patient's tumor also had a 1q-arm gain. Meanwhile, the DTCs from this patient harbored all the clonal copy number aberrations, though not the subclonal gain or any DTC-specific copy number changes.

For the second patient, the DTCs shared all the clonal CNAs of the primary tumor as well as its subclonal gains.

When they examined the DTCs from the patient with the lymph node metastasis, the researchers found a more complex progression model. This patient's DTCs originated from multiple subclones of the lymph node metastasis. Further, the DTCs collected three years after diagnosis had all the clonal aberrations found in the lymph node metastasis, plus a few more, suggesting that they descended from a subclone of the lymph node metastasis, survived therapy, and continued to evolve.

All in all, the researchers said these findings support the model of late dissemination of breast cancer cells to the bone marrow.

"While our sample size is small and further study is needed, these results raise the hope that early detection strategies can lead to diagnosis and treatment before the emergence of such metastatic clones," the authors wrote in their paper.