scholarly journals Detection of clonal CD34+19+ progenitors in bone marrow of BCL2-IgH- positive follicular lymphoma patients

Blood ◽  
1995 ◽  
Vol 86 (12) ◽  
pp. 4691-4698 ◽  
Author(s):  
EA Macintyre ◽  
C Belanger ◽  
C Debert ◽  
D Canioni ◽  
AG Turhan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Sorting ◽  
Chain Reaction ◽  
Selection Procedures ◽  
Polymerase Chain ◽  
B Cell Precursors ◽  
Selection Of

The frequent occurrence of BCL2-IgH rearrangements in follicular lymphoma (FL) makes detection of low numbers of tumor cells possible by polymerase chain reaction (PCR). The presence of BCL2-IgH in the bone marrow (BM) and peripheral blood of many FL patients at the time of autografting has led to the suggestion that selection of the CD34- enriched fraction may lead to reinfusion of lower numbers of tumor cells. To address this issue, we PCR-amplified BCL2-IgH from fluorescence-activated cell sorting (FACS)-purified BM CD34+ and CD34- fractions in seven FL patients showing a PCR-detectable translocation in the major breakpoint region of BCL2, five of which showed morphological BM involvement. The total CD34+ fraction showed diminished but residual positivity in the first two cases tested. Therefore, BM cells from the remaining five patients were sorted for the CD34+19- immature population, the CD34+19+ B-cell precursors, and the CD34–19+ mature B-cell fraction. The CD34+19- subpopulation was negative in four of five, despite evident BM infiltration in three cases. In contrast, the CD34+19+ fraction was positive in all three cases tested. These cells represented 0% to 50% (mean, 18%) of the total CD34+ population, suggesting that, if reinfusion of BCL2-IgH- positive cells plays a role in postautograft relapse in FL, therapeutic CD34 selection procedures should include additional purging of the CD34+19+ B-cell precursors or, at least, assessment of the proportion of CD19+ cells in the CD34+ fraction and its correlation with clinical outcome postreinfusion.

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction.

1994 ◽  
Vol 12 (4) ◽  
pp. 725-729 ◽  
Author(s):  
M Gerhard ◽  
H Juhl ◽  
H Kalthoff ◽  
H W Schreiber ◽  
C Wagener ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Tumor Cells ◽  
Normal Bone Marrow ◽  
Specific Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Normal Bone ◽  
Polymerase Chain

PURPOSE To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas. PATIENTS AND METHODS A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay. RESULTS In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method. CONCLUSION Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.

scholarly journals Application of long-distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B- cell neoplasms

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 985-994 ◽  
Author(s):  
T Akasaka ◽  
M Muramatsu ◽  
H Ohno ◽  
I Miura ◽  
E Tatsumi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cell ◽  
Tumor Cells ◽  
Chromosomal Translocation ◽  
Pcr Amplification ◽  
Chromosomal Translocations ◽  
Chain Reaction ◽  
Long Distance ◽  
Neoplastic Cells ◽  
Polymerase Chain

Abstract Junctional sequences created by chromosomal translocations in mature B- cell neoplasms, which involve immunoglobulin gene loci (IG) and putative proto-oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation, we have developed a novel strategy based on long-distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA was extracted from tumor cells carrying t(14;19)(q32;q13), t(8;14)(q24;q32), t(3;22)(q27;q11), t(2;3)(p12;q27), or t(3;14)(q27;q32). Thirty-two to 35-mer oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to IG constant region genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma, and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5′-BCL6/C mu, and 5′-BCL6/C gamma for t(3;14). In Burkitt's lymphoma/leukemia, all materials in which c- MYC rearrangements were detectable by conventional Southern blot hybridization showed positive LD-PCR amplification. The sizes of the amplified fragments varied from 1.8 kb to 12 kb, and these were specific to each material. Serial dilution of tumor cells or DNA in negative materials demonstrated a single band on agarose gel electrophoresis stained with ethidium bromide at a level of sensitivity of 10(-3), and hybridization with radioactive probe improved the level by one order of magnitude (1 cell in 10(4)), indicating that this LD- PCR approach is a sensitive technique capable of detecting minimal residual disease. Thus, the present study provided a useful tool for diagnosis and subsequent management of B-cell neoplasms characterized by specific chromosomal translocations.

Detection of tumor cells in purged bone marrow and peripheral-blood mononuclear cells by polymerase chain reaction amplification of bcl-2 translocations.

1994 ◽  
Vol 12 (5) ◽  
pp. 1021-1027 ◽  
Author(s):  
R S Negrin ◽  
J Pesando
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Before And After ◽  
Polymerase Chain

PURPOSE To compare bone marrow (BM) before and after purging with monoclonal antibodies (MAbs) and complement with peripheral-blood mononuclear cells (PBMNCs) for tumor-cell contamination by amplification of t(14;18) sequences using the polymerase chain reaction (PCR). PATIENTS AND METHODS Sixty patients with non-Hodgkin's lymphoma (NHL) undergoing autologous BM transplantation were evaluated. Six BM biopsies were performed at the time of harvesting and evaluated morphologically for tumor involvement. The harvested BM was treated with a panel of anti-B-cell MAbs directed against CD9, CD10, CD19, and CD20, followed by rabbit complement. Clonogenic assays were performed before and after purging. DNA was extracted and t(14;18) sequences amplified by PCR. PBMNCs collected by apheresis for back-up purposes were similarly evaluated. RESULTS Fifteen patients (25%) were PCR-positive before BM purging. Following MAb- and complement-mediated purging, there was a reduction in the PCR-amplified signal in 10 patients (67%). There was no reduction in colony-forming unit granulocyte-macrophage (CFU-GM) colony growth following purging. Eight of these 15 patients (53%) had morphologic evidence of BM involvement at the time of harvesting. In these eight patients, only three had a reduction in the PCR-amplified products, as compared with all seven who were morphologically negative at the time of BM harvesting (P = .026). Fourteen of these 15 patients had PBMNCs collected near the time of BM harvesting and 12 (86%) were PCR-positive. CONCLUSION BM purging with MAbs and complement results in reduction in the number of t(14;18)-positive tumor cells, especially in those patients who have no morphologic evidence of BM disease at the time of harvesting. Purged BM was less contaminated with t(14;18)-positive cells than unpurged PBMNCs, which were frequently contaminated with tumor cells.

Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.

1997 ◽  
Vol 15 (7) ◽  
pp. 2701-2708 ◽  
Author(s):  
A Zippelius ◽  
P Kufer ◽  
G Honold ◽  
M W Köllermann ◽  
R Oberneder ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Tumor Cells ◽  
Carcinoma Cells ◽  
Cell Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

PURPOSE This study was designed to evaluate the potential of reverse-transcriptase polymerase chain reaction (RT-PCR) analyses for the detection of micrometastatic carcinoma cells in bone marrow (BM). PATIENTS AND METHODS The specificity of RT-PCR assays with primers specific for various tumor-associated and organ-specific mRNA species was examined by analysis of 53 BM aspirates from control patients with no epithelial malignancy. In addition, BM samples from 63 patients with prostate cancer (n = 53) or breast cancer (n = 10) were analyzed by RT-PCR with primers specific for prostate-specific antigen (PSA) mRNA. As a reference method, all samples were analyzed simultaneously by an established immunocytochemical assay, using monoclonal antibodies (mAbs) against cytokeratins (CK) for tumor-cell detection. RESULTS Seven of eight marker species could be detected in a considerable number of BM samples from control patients: epithelial glycoprotein-40 (EGP-40; 53 of 53 samples), desmoplakin I (DPI I; five of five), carcinoembryonic antigen (CEA; five of 19), erb-B2 (five of seven), erb-B3 (six of seven), prostate-specific membrane antigen (PSM; four of nine), and CK18 (five of seven). Only PSA mRNA was not detected in any of the 53 control BM samples. In serial dilution experiments, the PSA RT-PCR assay was able to detect five LNCaP prostate carcinoma cells in 4 x 10(6) BM cells. CK-positive cells were found in 20 patients (37.7%) with prostate cancer, while PSA mRNA was found in only 15 (28.3%; P = .04). Moreover, despite the recent observation that PSA is also expressed in mammary carcinomas, none of the 10 CK-positive BM samples were PSA mRNA-positive. CONCLUSION Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

The Use of Archival Bone Marrow Specimens in Detecting B-Cell Non-Hodgkin's Lymphomas Using Polymerase Chain Reaction Methods

2000 ◽  
Vol 36 (3-4) ◽  
pp. 347-352 ◽  
Author(s):  
Tetsuro Nagasaka ◽  
Raymond Lai ◽  
Yuan-Yuan Chen ◽  
Wengang Chen ◽  
Daniel A. Arber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
B Cell ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Hodgkin's Lymphomas

scholarly journals Rearrangement and selection of VH11 in the Ly-1 B cell lineage.

1990 ◽  
Vol 172 (1) ◽  
pp. 371-374 ◽  
Author(s):  
C E Carmack ◽  
S A Shinton ◽  
K Hayakawa ◽  
R R Hardy
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cells ◽  
B Cell ◽  
Peritoneal Cavity ◽  
Cell Lineage ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Vh Genes ◽  
Self Antigen ◽  
Selection Of

One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.

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