Colon Neoplastic Cells Do Not Originate from Bone Marrow-derived Cells after Sex-mismatched Bone Marrow Transplantation

Materials and Methods

Samples. All samples were collected at Keio University Hospital. Written informed consent was obtained from each patient after explanation of the nature and possible consequences of the study. The local Ethics Committee approved the study.

Case 1. When the patient, a 62-year-old woman, was 47 years old (1992), she received a diagnosis of acute promyelocytic leukaemia (APL); therefore, she was treated with all-trans retinoic acid (ATRA) and systemic chemotherapy [two courses of behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine plus prednisolone, six courses of vincristine, endoxan, methotrexate plus prednisone and one course of behenoyl cytosine arabinoside, daunorubicin, 6-mercaptopurine, prednisolone plus daunomycin]. Following recognition of her second complete response (CR) to chemotherapy, she underwent allogeneic BM transplantation [Human Leukocyte Antigen and ABO total match, blood type A/Rh+] on January 17, 1995. The donor was her elder brother, who was 58 years old at the time. On February 13, 1995, a BM puncture examination was used to confirm that the BM cells were donor-derived cells (46,XY). Graft versus host disease (GVHD) was tightly controlled by medications, and she was discharged from the hospital on March 12, 1995. Following discharge, she was treated as an outpatient. On June 20, 2007, she underwent a colonic endoscopy following a positive faecal occult blood test. The endoscopy revealed that she had a flat elevation, approximately 10 mm in diameter, in the sigmoidal colon (Figure 1A); therefore, she underwent EMR to remove this lesion. The pathological examination revealed a well-differentiated tubular adenocarcinoma within an adenoma (int, INFα, m, ly0, v0) (Figure 1B).

Case 2. When the patient, a 42-year-old woman, was 28 years old (1994), she received a diagnosis of APL. She underwent ATRA and systemic chemotherapy, comprising of four courses of consolidation therapy and five courses of maintenance therapy, prescribed by the JALSG AML87 protocol (10). Following recognition of her second CR, she underwent allogeneic BM transplantation (HLA and ABO total match, blood type O/Rh+) on March 19, 1996. The donor was her elder brother, who was 44 years old at the time. On April 30, 1996, a BM puncture examination was used to confirm that BM cells were donor-derived cells (46,XY). Graft versus host disease (GVHD) was tightly controlled, and she was discharged from the hospital on July 31, 1996. Following discharge she was treated as an outpatient. On December 19, 2007, she underwent a colonic endoscopy following a positive faecal occult blood test. There were two polyps, one in each arm of her ascending colon; the oral-side polyp was about 3 mm, and the anal-side polyp was about 10 mm (Figure 2A and C). Therefore, she underwent EMR; pathological examination revealed that the oral-side polyp was a tubular adenoma with severe dysplasia (Figure 2B) and the anal-side polyp contained a well-differentiated tubular adenocarcinoma within an adenoma (int, INFβ, med, pM, ly0, v0) (Figure 2D).

FISH (11). We analysed the histological and pathological samples using FISH and probes for mammalian X and Y chromosomes. Haematoxylin-eosin-stained colonic tissue sections from both patients described above, who had undergone BM transplantation, were examined. We used a digoxigenin-labelled FISH probe for human Y chromosome and Cy5-labelled FISH probe for rat X chromosome. Following the FISH analysis, the samples were subjected to immunostaining to label for cytokeratin and CD45 to confirm that the cells with Y-positive signals were epithelial cells and not haemocytes.

Analysis of X/Y chromosomes. After removing paraffin from the tissue samples, the samples were soaked in phosphate buffered saline (PBS) for about 5 min, and then treated with protease (0.1% pepsin/0.1 M HCl) for about 3 min. After these reactions, the samples were washed in PBS, and dehydrated in a series of alcohol solutions. Digoxigenin-labelled Y-chromosome and Cy5-labelled X-chromosome FISH probes were applied to the pepsin-treated samples, and the probes were treated simultaneously on a hot plate (80°C) for 10 min; probes hybridized to targets within the sample, overnight at 37°C. Tissue samples were stringently washed, the digoxigenin label was then labelled with anti-digoxigenin, and probe signals were analysed.

Immunostaining (CD45, Pan-cytokeratin). Samples that were subjected to FISH analysis were then blocked using PBS with 5% milk and 0.3% Triton X-100. We diluted primary antibodies (FLEX Monoclonal Mouse Anti-Human CD45, Leucocyte Common Antigen and FLEX Monoclonal Mouse Anti-Human Cytokeratin; Dako North America, Inc. Carpinteria, California, USA) in PBS with 5% milk and 0.05% Tween20, which reacted to anti-digoxigenin-Cy5 for 1 h in 37°C. Samples were then washed in PBS for 15 min, and secondary antibodies (anti-rabbit-Alexa488 and anti-mouse-Alexa594; Life Technologies, Carlsbad, California, USA), diluted at 1/500, were then incubated with the samples for 30 min. Samples were washed in PBS for 15 min, then fixed with 4% paraformaldehyde in PBS for 5 min, and washed in PBS. Finally, samples were stained with DAPI (4’,6-Diamidino-2-phenylindole dihydrochloride; DOJINDO Laboratories, Kumamoto, Japan).

Microscopic observation. Samples were mounted on slides using mounting medium that included an anti-bleaching agent. Signals from FISH probes and data were analysed using the Leica CW-4000 system (Leica Microsystems GmbH, Wetzlar, Germany). We collected and merged 10 images of probe signals by moving the microscopical stage by 0.5 μm in depth on the sections, combining the signal by depth from the upper side to the lower side of sections into one image.