Detection and quantitation of malignant cells in the peripheral blood of multiple myeloma patients

Abstract

One of the distinguishing features of multiple myeloma (MM) is the proliferation of plasma cells that home to the bone marrow (BM). However, there still remains some uncertainty concerning the presence of related malignant cells in the peripheral blood of myeloma patients. Using consensus oligonucleotide primers, we amplified the third complementary determining region (CDR3) of rearranged immunoglobulin heavy chain alleles from MM marrow samples by polymerase chain reaction (PCR). From the sequences of the products, we derived allele-specific oligonucleotides (ASO), and these were used in subsequent amplification reactions to detect malignant clones in the peripheral blood of myeloma patients. This method is highly specific and sensitive to 1 malignant cell in the background of 10(5) normal cells. Using this method we detected circulating malignant cells in 13 of 14 previously untreated MM patients. Furthermore, by applying ASO-PCR to artificial titrations of initial BM DNA sample into normal peripheral blood lymphocyte (PBL) DNA we were able to generate standard curves and quantitate the amount of tumor in the patient PBL. We observed a wide variation in the amount of circulating tumor between patients. In addition, we found that the incidence of circulating tumor cells was independent of BM tumor burden and stage of disease. The detection and quantitation of circulating tumor cells in the PBL of MM patients may offer an alternative assessment of the disease and may be an important consideration in the use of peripheral stem cells in bone marrow transplantation.

CLLU1 expression levels predict time to initiation of therapy and overall survival in chronic lymphocytic leukemia

“…cDNA was amplified with specific upstream primer corresponding to variable region (VH) family specific primer VH1 through VH6 that anneal to sequences in the leader region (27) along with a C μ or C γ primer (28). In cases where such amplification failed, primers specific to framework I region and the heavy‐chain joining region ( J H ) were used (29). RT‐PCR products were purified and sequenced from both strands.…”

Evaluation of minimal residual disease in multiple myeloma patients by fluorescent‐polymerase chain reaction: the prognostic impact of achieving molecular response

“…If this happens, the primer and/or probe will be mismatched and PCR efficiency will be dramatically reduced (Gonzalez et al , 2003). Finally, molecular tests based on ASO‐PCR of IGH are methodologically complex, laborious and expensive and therefore difficult to apply as a routine MM patient follow‐up technique (Billadeau et al , 1992; Raab et al , 2005; Sarasquete et al , 2005). Other studies based on fluorescent (F)‐PCR of IGH have been unable to add any prognostic information (Davies et al , 2001).…”

Minimal residual disease detection in lymphoma and multiple myeloma: impact on therapeutic paradigms

“…One of the major technical advances in MRD detection in lymphoid tumors, using both tumor‐specific translocations and the IGHV rearrangement, has been the development of quantitative‐PCR tools (Figure ). Although preliminary efforts were made in the 1990s using limiting dilution and end point product quantification tools , it was the development of TaqMan‐based approaches that allowed the widespread use of quantitative PCR in the context of large clinical studies. Currently, most studies do include MRD detection by real‐time quantitative PCR (RQ‐PCR) .…”

“…Despite its complexity, several reports have used MRD for MM (Figure ), and a considerable amount of knowledge has been documented over time (Table ), leading to a number of clinically relevant assessments: Molecular remission is highly uncommon in patients receiving treatment based exclusively on cytotoxic chemotherapy, even if delivered at high doses in the context of ASCT‐based programs . Whenever PCR negativity occurs, it is always a transient finding . On the other hand, persistent long‐term MR can be achieved in patients receiving conventional allogeneic transplantation, indicating that the intrinsic chemoresistance of malignant plasma cells can be overcome using alternative cytotoxic mechanisms . The use of quantitative PCR (either RQ‐PCR or the less sophisticated approaches such as limiting dilution PCR ) allows effective outcome discrimination, with a clear adverse prognosis for patients with higher residual tumor burden . Patients receiving consolidation therapy based on new drugs after ASCT experience further major tumor burden reduction, as assessed by RQ‐PCR, and in some cases enter a prolonged MR that appears, in most respects, similar to that achieved with allogeneic transplantation .…”