A Sensitive Molecular Method for Detection of Disseminating Hematopoietic Tumor Cells by Using Specific Epigenetic DNA Methylation Biomarkers

Background: Most cancer deaths are caused by hematogenous spread and subsequent metastasis. Emerging data indicates that the presence of circulating tumor cells in peripheral blood (CTC) and disseminating tumor cells in bone marrow (DTC) is an early event in tumorigenesis. These circulating or disseminating rare tumor cells may represent a distinct clone with cancer stem cell properties (metastatic stem cells). Clinically, the presence of CTC and DTC is relevant to overt metastases. Early detection and characterization of these rare biologically distinct tumor cells with sensitive and specific methods provide vital information for cancer biology and early clinical intervention and thus improve patients’ survival.

Method Design: Genomic DNA was extracted from patient blood, bone marrow aspirate specimens and cancer cell lines prior to digestion with 4 methylation sensitive restriction enzymes. Hypermethylated CpG island regions in tumors, in contrast to their counterpart in normal cells which are completely digested, remain resistant to digestion and therefore can be differentially amplified by PCR. Thus, the specific PCR products on the gel represent the specific methylation loci in the tumor cells in the patient specimens.

Results: First, we tested a total of 26 cancer cell lines including 17 hematopoietic tumors and 9 solid tumors. The hematopoietic tumor cell lines represent a spectrum of B-cell malignancies, T-cell lymphoblastic leukemia and acute myeloid leukemia. The solid tumor cell lines represent the major anatomic sites of human carcinoma including lung, colon, breast, prostate, ovarian and skin (melanoma). We found multiple DNA methylation markers specific for lymphoid, myeloid and solid tumors. By using DLC-1 (deleted in liver cancer-1), a tumor suppressor gene, as a single marker, CpG island methylation was detected in 13 out of 17 hematopoietic tumor cell lines (76%) and 6 out of 9 solid tumor cell lines (67%). We then used B-cell acute lymphoblastic leukemia (B-ALL) as a testing case to verify the method in a total of 135 clinical specimens. DLC-1 methylation was detected in 64% and 54% of diagnostic bone marrow aspirates and peripheral blood specimens, respectively, but none was detected in normal or non-leukemic bone marrow or blood control samples. By adding two additional methylation markers PCDHGA12 and CDH1, greater than 90% of B-ALL patients can be detected. We also traced 4 B-ALL cases and 4 follicular lymphoma cases up to 10 years retrospectively and found that the DLC-1 methylation is exactly correlated with patient clinical status. Lastly, by mixing normal and leukemic cell genomic DNA, analytic sensitivity was determined as 0.1% or 10⁻³ in a single-step PCR and 0.00001% or 10⁻⁶ in a nested PCR suggesting that this method is capable of detecting as few as 5 leukemic cells in a single-step PCR.

Conclusion: By utilizing tumor specific DNA methylation marker(s), we have developed a simple, highly sensitive and specific gel-based PCR assay, namely Multiple Methylation Sensitive Enzyme Restriction PCR (MSR-PCR) for detection of CTC and DTC from blood and bone marrow of majority of hematopoietic malignancies. The method can potentially be used for early diagnosis and molecular monitoring in vast majority of clinical cancer patients.