Funding for this study was provided in part by an unrestricted gift from the Amway Corporation. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: JP GL JC. Performed the experiments: VK NS MS DC GL ED RL JP. Analyzed the data: JP GL RS JC RL. Contributed reagents/materials/analysis tools: JC GL JP NS. Wrote the paper: JP JC.
Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically.
We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT), using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes.
All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event.
Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome.
Tumor cell fusion with motile leucocytes such as myeloid lineage cells or stem cells has been put forward as a unifying explanation for metastasis
However, fusion and genomic hybridization have yet to be proven on a genetic basis in human cancer, since genomic differences between cells from the same patient cannot be readily distinguished. To circumvent this problem we have analyzed secondary malignancies arising post allogeneic bone marrow transplants (BMT). In two previous reports we demonstrated donor alleles in patient cancer cells, but there was no provision to identify patient alleles and fusion could thus not be proven
All samples used in this study were preexisting and de-identified before being received by the Yale research team. Exemption was granted under Yale IRB protocol #070900309 (JP and RL) from the Yale University Human Research Protection Program, Institutional Review Board.
The patient was a 68-year-old man who received an allogeneic BMT from his brother for treatment of B-cell lymphoma. His last engraftment/chimerism profile was Recipient 3%; Donor 97%. Six years later the patient was diagnosed with metastatic melanoma involving lymph nodes, liver and brain, derived from an unknown primary tumor. We analyzed a brain metastasis (designated “MH3”) consisting of a 0.5×0.2×0.3 cm formalin-fixed paraffin-embedded (FFPE) tissue. The tumor was surgically removed, fixed in formalin, and embedded in paraffin by standard histological procedures. Pre-transplant donor and patient lymphocytes were stored at −90°C in the Yale-New Haven Hospital Stem Cell Bank and retrieved after the tumor analyses were completed.
Handling and processing of tissue samples was carried out using ultraclean, DNA-free equipment. Five μ-thick histological sections were cut and immunostained for LCA/CD45 (clone 2B11+PD7/26, Dako, catalog N1514) using an autostainer (DAKO, Carpinteria, CA) at the Yale Dermatopathology Laboratories. The antibody was tested for staining efficiency as described in
DNA extraction and STR analyses were by the Denver Police Department Crime Laboratory DNA Unit using standard forensic operating and valided procedures
PCR was performed with the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, Carlsbad, CA, USA). Generally, 1 ng of total DNA was targeted in each PCR amplification. In samples with less DNA (<0.1 ng/µl), samples were concentrated 5–20 fold using a Microcon centrifugal filter (Ultracel Ym-100, Millipore, Billerica, MA, USA).
Each STR locus was selected to be neutral with respect to other genetic linkage or associations with either Mendelian or non-Mendelian disorders. The loci were polymorphic and exhibited acceptable levels of heterozygosity, typically 70% or higher. They could be assayed together as a PCR multiplex and were robust for degraded DNA
Allele signal peaks may overlap technical “stutter” positions from other alleles. Validation studies have established interpretation guidelines for forensic markers to distinguish true allele signals from stutter for any given allele at any locus
Bayesian statistical models of fusion and donor cell contamination were fitted and compared using Markov chain Monte Carlo methods
We performed extensive pathology analyses to ensure that laser dissected tumor samples were not contaminated by infiltrating donor leukocytes. To detect leucocytes we used an antibody to leucocyte common antigen (LCA/CD45), expressed on the surface of mature leukocytes and hemopoietic progenitor cells. In positive control experiments the antibody showed >99% staining efficiency against test cases of dermatitis and lymphoma (Figs. S1 and S2, Table S1 in
Staining of the MH3 melanoma for LCA/CD45 revealed that some regions contained LCA/CD45-positive leucocytes intermixed with LCA/CD45-negative melanoma cells (
A. An area with brown LCA/CD45-positive leucocytes (arrow) intermixed with blue LCA/CD45-negative cancer cells. B-D. Adjacent areas from the same section containing only blue LCA/CD45-negative cancer cells.
A. An area of S100-positive tumor cells admixed with infiltrating S100-negative leucocytes (arrows). B. An area containing only S100-positive tumor cells. More detailed pathology analyses are presented in Figs S3 and S4 in
Tumor sections were stained with LCA/CD45 prior to laser dissection and tumor cells were dissected free of LCA/CD45-positive leucocytes. Tumor cells were isolated from 9 regions throughout the tumor (samples 1–9) and DNA was extracted and amplified for alleles at 14 STR loci. All alleles found in the pre-transplant donor and patient blood lymphocytes were also detected in tumor cells. For each locus there was at least one allele common to both the donor and patient, consistent with the fraternal relationship. Eight loci exhibited donor- specific alleles and six of these exhibited both donor and patient specific-alleles. This indicated that the tumor cells were donor-patient hybrids (
Shown are “informative” loci exhibiting donor and patient specific alleles in pre-BMT lymphocytes. Tumor loci are listed in order of relative abundance of the donor-specific alleles (red asterisk) compared to patient-specific (blue asterisk) and shared alleles (black asterisk). Allele peaks <50 relative fluorescence units were censored as “no call” (open circles). Loci with no detectable alleles after PCR amplification (—).
Locus | Chromosome | Donor |
Patient |
Tumor |
Tumor Genotype |
D13S317 | 13 | 9,11 | 9,10 | 9,11 | D/S |
D19S433 | 19 | 12,13 | 13,14 | 12,13,14 | D/P/S |
CSF1PO | 5 | 10,11 | 11,12 | 10,11,12 | D/P/S |
D16S539 | 16 | 9,11 | 9,13 | 9,11,13 | D/P/S |
FGA | 4 | 20,22 | 20,21 | 20,21,22 | D/P/S |
D7S820 | 7 | 8,9 | 8,12 | 8,9,12 | D/P/S |
D8S1179 | 8 | 13,14 | 13,13 | 13,14 | D/S |
vWA | 12 | 19,20 | 19,19 | 19,20 | D/S |
STR units: number of tandem repeats of the locus-specific tetranucleotide sequence. The X and Y chromosomes were detected by the amelogenin assay
Locus | Chromosome | Donor |
Patient |
Tumor |
Tumor Genotype |
n.a. | X,Y | X,Y | X,Y | X,Y | X,Y |
D18S51 | 14 | 14,14 | 14,20 | 14,20 | P/S |
TH01 | 11 | 7.9.3 | 7.9.3 | 7.9.3 | S/S |
D21S11 | 2 | 28,29 | 28,29 | 28,29 | S/S |
D2S1338 | 2 | 20,26 | 20,26 | 20,26 | S/S |
TPOX | 2 | 8,8 | 8,8 | 8,8 | S/S |
D5S818 | 5 | 12,13 | 12,13 | 12,13 | S/S |
STR units: number of tandem repeats of the locus-specific tetranucleotide sequence. The X and Y chromosomes were detected by the amelogenin assay
Finally, we fit statistical models to compare the likelihood of donor BMDC-tumor cell fusion versus donor leucocyte contamination (
Panels A and B: Deviances under contamination and fusion models; smaller deviances indicate better fit. Panels C and D: A calibration procedure shows the observed LPML difference (red lines) is rare under the contamination model but typical under the fusion model. More detailed statistical analyses are presented in
STR analyses of the tumor DNA revealed that donor and patient alleles were present together at multiple loci and that there were widespread allelic imbalances and aneuploidy. One drawback was the inability to perform STR analyses on individual tumor cells, but for this tumor the lower limit for DNA recovery for STR analyses was about 500 cells, even using the DNA extraction procedure for FFPE cells that improved DNA recovery. Thus, we could not definitively rule out that the patterns might be due to a chimeric mixture of patient tumor cells and donor BMDCs. However this is unlikely for the following reasons: 1) For a given locus the allelic ratios were similar throughout the tumor. It is difficult to explain these repeating patterns as due to chimeric mixtures as this would require that the different cell types existed together in the same ratios throughout the tumor. Notably, while most of the informative tumor loci had patient and shared alleles in greater relative abundance to donor-specific alleles, tumor locus D13S317 was reversed, with the patient-specific allele absent and the donor-specific allele in prominence. Since the initial dose of genomic DNA for each sample was determinative of PCR product intensity and varied widely between samples, given the consistency in allelic ratios from sample to sample the reversal of DNA dosage at locus D13S317 cannot be explained by preferential PCR or leukocyte contamination. 2) The tumor cells were dissected from regions free of LCA-positive cells (
In stem cell biology both transdifferentiation and fusion appear to be operative in the transformation of stem cells into differentiated somatic cells
In earlier studies, experimental tumor hybrids generated
A model for metastasis resulting from fusion of bone marrow-derived cells is diagramed schematically in
A motile BMDC (red) such as a macrophage or stem cell is drawn to a cancer cell (blue). The outer cell membranes of the two cells become attached. Fusion occurs with the formation of a bi-nucleated heterokaryon having a nucleus from each of the fusion partners. The heterokaryon goes through genomic hybridization creating a melanoma-BMDC hybrid with co-expressed epigenomes, conferring deregulated cell division and metastatic competence to the hybrid.
Figures S1, S2, S3, S4, S5 and Table S1.
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Figures S6, S7, S8, S9 and Table S2.
(PDF)
We thank Prof. Douglas Brash, Yale School of Medicine, for first suggesting the use of second malignancies following BMT and for his help with the manuscript.