Multiple mutation analyses in single tumor cells with improved whole genome amplification

Abstract

Combining whole genome amplification (WGA) methods with novel laser-based microdissection techniques has made it possible to exploit recent progress in molecular knowledge of cancer development and progression. However, WGA of one or a few cells has not yet been optimized and systematically evaluated for samples routinely processed in tumor pathology. We therefore studied the value of established WGA protocols in comparison to an improved PEP (I-PEP) PCR method in defined numbers of flow-sorted and microdissected tumor cells obtained both from frozen as well as formalin-fixed and paraffin-embedded tissue sections. In addition, the feasibility of I-PEP-PCR for mutation analysis was tested using clusters of 50-100 unfixed tumor cells obtained by touch preparation of ten breast carcinomas by conventional sequencing of exon 7 and 8 of the p53 gene. Finally, immunocytochemically stained microdissected single disseminated tumor cells from bone marrow aspirates were investigated with respect to mutations in codon 12 of Ki-ras by restriction fragment length polymorphism (RFLP)-PCR after I-PEP-PCR. The modified I-PEP-PCR protocol was superior to the original PEP-PCR and DOP-PCR protocols concerning amplification of DNA from one cell (efficiency rate I-PEP-PCR 40% versus PEP-PCR 15% and DOP-PCR 30%) and five cells (efficiency rate I-PEP-PCR 100% versus PEP-PCR 33% and DOP-PCR 20%). Preamplification by I-PEP allowed 100% sequence accuracy in > 4000 sequenced base pairs and Ki-ras mutation detection in isolated single disseminated tumor cells. For reliable microsatellite analysis of I-PEP-preamplified DNA, at least 10 unfixed cells from fluorescence-activated cell sorting, 10 cells from frozen tissue, or at least 30 cells from formalin-fixed and paraffin-embedded tissue sections were required. Thus, I-PEP-PCR allowed multiple reliable microsatellite analyses suited for microsatellite instability and losses of heterozygosity and mutation analysis even at the single cell level, rendering this technique a powerful new tool for molecular analyses in diagnostic and experimental tumor pathology.

Figure 1.

Efficiency of I-PEP-PCR compared to unmodified PEP-PCR and DOP-PCR. A: SW480 cells were sorted as single, five, ten, and 100 cells by FACS in ten parallel samples, respectively. The cells were lysed and DNA was preamplified either by I-PEP, PEP-PCR or DOP-PCR. A 1/30th aliquot was used for specific amplification of a 540-bp β-globin fragment. Positive controls were 10 ng SW480 genomic DNA preamplified (first +) and nonpreamplified (second +). Negative controls (−) were H₂O in preamplification PCR followed by specific PCR. PCR products were analyzed by ethidiumbromide-stained 2.5% agarose gels. B: Cell clusters consisting of 50, 100, 200, 500, and 1000 cells microdissected from methylene blue stained frozen bladder tissue sections were lysed and preamplified by I-PEP-PCR and PEP-PCR, respectively. One-tenth DNA aliquots were used for specific amplification of a 540-bp β-globin fragment. PCR products were analyzed by ethidiumbromide-stained 2.5% agarose gels. C: Cell clusters consisting of 30, 50, 100, 250, 500, and 1000 cells were microdissected from a H&E stained formalin-fixed and paraffin-embedded colonic tissue section, lysed and preamplified according to I-PEP-PCR and PEP-PCR protocol, respectively. One-tenth aliquots were used for specific amplification of TP53 PCR microsatellite marker (120-bp fragment, uninformative alleles). As positive controls a cell cluster of > 1000 cells was used. PCR products were analyzed by silver-stained denaturating polyacrylamide/urea gels.

Figure 2.

Microsatellite analysis by silver-stained denaturating polyacrylamide/urea gels. A: SW480 cells were sorted into samples of 1, 5, 10, or 100 cells by FACS in ten parallel samples, respectively. The cells were lysed enzymatically and DNA was preamplified by I-PEP-PCR. A 1/30th aliquot was used for amplification of D2S123 microsatellite marker (200- to 230-bp fragments). B: Methylene blue-stained and microdissected cell clusters of 10, 25, and 50 cells from frozen tissue were preamplified either by I-PEP-PCR (top) or PEP-PCR (bottom) and a 1/12th aliquot was used for amplification of D2S123 microsatellite marker. C: Microdissected cell clusters of 30, 50, 100, and 250 cells from H&E stained sections from formalin-fixed and paraffin-embedded tissue (right) were preamplified either by I-PEP-PCR (upper part) or PEP-PCR (lower part) and a 1/12th aliquot was used for amplification of D2S123 microsatellite marker. D: Microdissected cell clusters of 100 cells from immunohistochemically p53-positive (p53 IHC+) and -negative (p53 IHC−) stained cells from sections of formalin-fixed and paraffin-embedded tissue were preamplified by I-PEP-PCR and a 1/12th aliquot was used for amplification of TP53ALS microsatellite marker.

Figure 3.

Sequence analysis of p53 gene after I-PEP-PCR. Tumor cell DNA from tumor touch preparations of breast cancer patients were preamplified by I-PEP-PCR. One-thirtieth aliquots were used for nested PCR amplifying exon 7 and exon 8 of p53 gene. PCR products were subsequently used for cycle sequencing. The positions of an 8-bp deletion (A; Del) and of mutated nucleotides (nt) are indicated (A-D). Mutated nucleotides are shown in italics.

Figure 4.

Ki-ras mutation analysis in isolated CK18-stained disseminated tumor cells in bone marrow. Microdissected cells were preamplified by I-PEP-PCR and 1/10th aliquot was used for subsequent RFLP-PCR. Undigested (157-bp fragments) and MvaI-digested DNA (143-bp fragments in mutated codon 12; 114-bp fragments in wild-type codon 12) are given in parallel for each sample (A, 1–11; B, 1–8). A: CK18 immuncytochemically positive stained cells were examined as single (Lanes 1, 2, 4, and 5) or few (5 < n < 10) cells (Lanes 3, 6, and 7). Lanes 1 and 2: cell samples from a pancreatic carcinoma patient. Lanes 3–7: cell samples from a rectum carcinoma patient. Lanes 8–11: negative controls (unstained hematopoietic cells, n ∼ 1000 each). B: Tumor tissue. Lanes 1–3: pancreatic carcinoma. Lane 4: normal pancreatic tissue. Lanes 5 and 6: rectum carcinoma. Lane 7: normal rectal tissue. Lane 8: CAPAN-1 cell line as positive control.