Novel real‐time PCR assay using a universal molecular marker for diagnosis of hematologic cancers

Dear Sir, Hematologic cancers, the third most common cause of cancer deaths, will kill 46,600 Americans this year. While most lymphomas and leukemias (LL), which consist of several types and subtypes, can be diagnosed by flow cytometric analysis of surface antigens, a large panel of markers (~50) is required. In 5–10% of cases, an initial diagnosis of malignancy is in doubt. In addition, detection of minimal residual disease (MRD) after chemotherapy is of major clinical importance as it may predict future recurrence. Routine MRD assays such as histology and flow cytometry are of low sensitivity, while PCR assays require identification of the specific form of LL and may need individualized assay conditions. Thus, a single assay for the diagnosis of all LLs and for the detection of MRD would be of major clinical benefit. Cytokines are secreted proteins that play a central role in the regulation of a wide array of cellular functions in the lymphohematopoietic system, including proliferation, differentiation and cell survival. Cytokines bind to and activate their cognate cell surface receptors. For certain cytokines, activated receptors recruit members of the Janus kinase (JAK) family of cytoplasmic tyrosine kinases, which in turn phosphorylate and activate transcription factors known as signal transducers and activators of transcription (STATs). Activated and dimerized STATs enter the nucleus and induce transcription of multiple genes having STAT recognition sites in their promoters. The pathway is negatively regulated by protein tyrosine phosphatases (SHP1) and by other proteins including SOCS1 and SYK, whose inactivation may result in up regulation. Upregulation of the JAK/STAT signaling pathway is frequent in LL. Epigenetic inactivation of tumor suppressor genes through aberrant methylation of their promoters is a very frequent event. While downregulation of expression of a candidate suppressor gene may be demonstrated by RNA or protein assays, these substances are labile and may not be of sufficient quality or quantity for detection in routine clinical samples. However, assays utilizing DNA (such as methylation-specific PCR [MSP]) are more robust and can be applied to a wide variety of samples, even those in alcohol fixatives. We initially determined the promoter methylation status of 3 negative regulators (SHP1, SOCS-1 and SYK) of JAK-STAT pathway in 90 LLs. using a standard PCR-based MSP assay. These initial analyses showed that SHP1 may be a near universal candidate marker for the diagnosis of malignancy. Thus, we developed a highly sensitive and specific semiquantitative real-time PCR assay for methylated SHP1 based on TaqMan probe technology. To develop real time PCR assay, LL samples (n 5 90) were obtained from the Flow Cytometry Facility of the Department of Pathology at the UT Southwestern Medical Center, Dallas, TX and its affiliated hospitals, after receiving Institutional Review Board permission. In brief, composition of the LL samples was as follows: 36 B-cell lymphoma (Burkitt, diffuse large B-cell, follicular, marginal zone and mantle cell lymphomas); 6 T-cell lymphomas (large cell anaplastic, peripheral T-cell); 36 lymphoid leukemias (acute and chronic); and 12 myeloid leukemias (acute and chronic). We also obtained 26 control samples (peripheral blood mononuclear cells [PBMC], lymph node or bone marrow) from healthy volunteers and hematologic patients without cancers. In addition, we used 12 Epstein-Barr virus transformed B-cell lymphoblastoid (BL) lines from the PBMCs of healthy individuals that had been in continuous culture for about 5 months when tested. To further validate and check the reproducibility of the real-time PCR assay, an additional 44 LL samples (27 B-cell lymphoma, 2 T-cell lymphoma, 9 acute lymphocytic leukemia [ALL], 6 acute myelogenous leukemia [AML]) were obtained from the Department of Pathology at the UT Southwestern Medical Center, Dallas, TX and its affiliated hospitals. We also obtained 60 control samples: 40 PBMC obtained from healthy volunteers and 20 bone marrows obtained from hematologic patients without cancers. Genomic DNA was extracted from cell lines, primary tumors and nonmalignant cells by using a DNA extraction kit (Serological Corporation, Norcross, GA). Aberrant promoter methylation of SHP1, SOCS1 and SYK was determined on bisulfite treated DNA by MSP, as reported by Herman and Baylin, using primers for methylated sequences and methodologies previously described for SHP1, SOCS1 and SYK. Primer sequences specific for the unmethylated form of the p16 gene were used to check the integrity of bisulfite-modified DNA from tumors. DNAs from peripheral blood lymphocytes (n 5 14) from healthy volunteers were used as negative controls for methylation specific assays. Genomic DNA, treated with Sss1 methylase (New England Biolabs, Beverly, MA) and