The differences and correlations of BCR‐ABL transcripts between peripheral blood and bone marrow assays are associated with the molecular responses in the bone marrow for chronic myelogenous leukemia

Previous studies concerning BCR‐ABL mRNA levels by quantitative real‐time RT‐PCR (Q‐PCR) for chronic myelogenous leukemia (CML) have shown a significant concordance between peripheral blood (PB) and bone marrow (BM) assays. The objective of this study was to determine whether molecular monitoring using PB was comparable to using BM for CML. A comparative study was performed that analyzed the Q‐PCR results of 712 simultaneous PB and BM samples from 330 patients before and during imatinib therapy. For the 78 paired pretreatment samples, the level of BCR‐ABL mRNA in PB was lower than that in BM (P = 0.007). Although the overall amounts of BCR‐ABL mRNA in the PB and BM were comparable (P= 0.072) and there was a strong correlation (r = 0.839, P < 0.001) with the 634 paired on‐treatment samples, the depth of the molecular response in PB was lower than that in BM (P < 0.001). The level of BCR‐ABL mRNA in PB was lower than that in BM where the BM BCR‐ABL mRNA < 1 log reduction (P < 0.001) or ≥ 1–< 2 log‐reductions (P = 0.008) from the baseline, and higher than that where the BM BCR‐ABL mRNA ≥ 2 log‐reductions (P < 0.001). A strong correlation (r = 0.811, P < 0.001) was only found where the BM BCR‐ABL mRNA < 1 log reduction. We conclude that the differences and correlations of BCR‐ABL mRNA between PB and BM assays depend on the depth of the molecular response in BM for CML during imatinib therapy. Am. J. Hematol., 2012. © 2012 Wiley Periodicals, Inc.     

Comparative analysis between RQ‐PCR and digital droplet PCR of BCL2/IGH gene rearrangement in the peripheral blood and bone marrow of early stage follicular lymphoma

BCL2/IGH rearrangements were analysed by polymerase chain reaction (PCR) at diagnosis in paired peripheral blood (PB) and bone marrow (BM) samples from 67 patients with stage I/II follicular lymphoma (FL). Real time quantitative PCR (RQ‐PCR) and digital droplet PCR (ddPCR) were performed in cases with a major breakpoint region (MBR+) at diagnosis and after localized radiotherapy and rituximab administration in order to investigate the applicability of ddPCR. The overall ddPCR/RQ‐PCR concordance was 81·9% (113/138 samples) and 97·5% in the 40/138 with quantifiable disease (RQ‐PCR≥10⁻⁵). At baseline, ddPCR allowed the recovery of a MBR+ marker in 8/18 (44·4%) samples that resulted MBR‐negative/minor cluster region‐negative/minor BCL2‐negative by qualitative PCR. Moreover, the tumour burden at diagnosis significantly predicted progression‐free survival (PSF) only when quantified by ddPCR. Paired PB and BM samples analysis demonstrated a high concordance in the detection of BCL2/IGH+ cells by qualitative and quantitative methods; in particular, 40/62 samples were positive by ddPCR (25 PB+/BM+; 9 PB+/BM−; 6 PB−/BM+), with 34/40 (85%) identified by the study of PB only. In conclusion, in localized FL, ddPCR is a promising tool for monitoring minimal residual disease (MRD) that is at least comparable to RQ‐PCR and potentially more accurate. PB is a suitable source for serial BCL2/IGH MRD assessments, regardless of the methodology utilized.     

Fluorescence In Situ Hybridization Monitoring of BCR-ABL-Positive Neutrophils in Chronic-Phase Chronic Myeloid Leukemia Patients during the Primary Stage of Imatinib Mesylate Therapy

We describe a method for monitoring chronic myeloid leukemia (CML) patients treated with imatinib that uses fluorescence in situ hybridization (FISH) to detect BCR-ABL in peripheral blood (PB) granulocytes. First, we compared this method, termed Neutrophil-FISH, with interphase FISH (i-FISH) analysis of bone marrow (BM), i-FISH analysis of PB mononuclear cells, and conventional cytogenetic analysis (CCA) of BM in 30 consecutive CML patients. We found the percentage of BCR-ABL-positive neutrophils as determined by Neutrophil-FISH to correlate best with the percentage of Philadelphia chromosome-positive metaphases in the BM determined by CCA (y = 0.8818x + 5.7249; r(2) = 0.968).We then performed a serial Neutrophil-FISH study of 10 chronic-phase CML patients treated with imatinib and found that the technique could clearly separate imatinib responders from nonresponders within 12 weeks of drug administration. There was a significant difference in the percentages of BCR-ABL-positive neutrophils between responder (mean 3 SD, 18.2% 3 11.8%) and nonresponder (82.4% 3 5.1%) groups at 12 weeks (P < .0001, Student t test).Together with real-time quantitative polymerase chain reaction analysis, Neutrophil-FISH represents another useful method for monitoring CML patients during the primary myelosuppressive stage of imatinib therapy because it is a quick, simple, and reliable method for assessing cytogenetic response.     

Quantitative PCR of bone marrow BCL2/IgH+ cells at diagnosis predicts treatment response and long-term outcome in follicular non-Hodgkin lymphoma

By real-time quantitative polymerase chain reaction (RQ-PCR), we evaluated BCL2/IgH(+) cells in the bone marrow (BM) and peripheral blood (PB) from 86 patients with follicular lymphoma treated with the sequential administration of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) and rituximab. At diagnosis, the amount of BCL2/IgH(+) cells in the BM was low (1 BCL2/IgH(+) cell in 1000-100 000 normal cells) in 43% of patients, intermediate (1 in 100-1000) in 34%, and high (> 1 in 100) in 23%. A 2 log decrease of BCL2/IgH(+) cells was achieved after CHOP and an additional 2 log reduction following rituximab. By multivariate analysis, a low level of BCL2/IgH(+) cells in the BM at diagnosis was the best predictor for the achievement of a complete clinical and molecular response. At 5 years, the event-free survival rates of patients with a low/intermediate or high tumor infiltration in the BM were 59% and 32%, respectively. The freedom from recurrence of patients who achieved a molecular response in the BM, no matter whether after CHOP alone or CHOP and rituximab, was 64% as compared to 32% for patients who did not (P < .006). RQ-PCR performed at presentation on BM samples predicts treatment response and long-term clinical outcome in patients with follicular lymphoma.     

The relative role of peripheral blood and bone marrow for monitoring molecular evidence of disease in follicular lymphoma by quantitative real-time polymerase chain reaction

Peripheral blood (PB) and bone marrow (BM) are used interchangeably for t(14;18) (IgH/BCL-2) molecular monitoring in follicular lymphoma (FL) and detection of rearrangement after treatment has been correlated to increased risk of relapse. To determine the relative value of each tissue, MBR t(14;18) was quantified by real-time polymerase chain reaction in 52 simultaneous paired PB and BM samples from 38 FL patients. In total, 79% of sample pairs taken in remission (n = 19) or when no morphological disease was evident in the BM (n = 29) had t(14;18) copy number within one log difference and the median difference was small. These findings suggest that, in remission, PB may be adequately monitored. In general, however, higher copy number was detected in BM than in the corresponding PB sample.     

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.     

Efficiency of interphase fluorescence in situ hybridization for BCR/ABL on peripheral blood smears for monitoring of CML patients: a comparison with bone marrow findings

Conventional cytogenetic analysis (CCA) is the standard method for monitoring of the Philadelphia (Ph) chromosome in chronic myeloid leukemia (CML). Evaluation of breakpoint cluster region/abelson murine leukemia (BCR/ABL) fusion using interphase fluorescence in situ hybridization on peripheral blood smears (PB-FISH) might be another approach allowing more frequent and less invasive follow-up investigations. Herein, BCR/ABL fusion gene was assessed on 21 PB smears from 16 CML patients in chronic phase. Results of PB-FISH were compared with those of CCA and interphase FISH on bone marrow aspirates (BM-FISH). PB-FISH analysis was combined with CD3 immunophenotyping that allowed simultaneous investigation of the leukemic status of CD3(+) T lymphocytes and scoring CD3(-) cells for BCR/ABL fusion gene. Moreover, the frequency of BCR/ABL fusion in nonlymphoid PB cells was estimated according to the differential leukocyte counts. The incidence of BCR/ABL(+) fusion signals in CD3(+) T cells of CML patients was 5.3% (SD +/- 1.9) and did not exceed the normal cut-off value of 8%. A significant correlation (P < 0.001) was found between results of PB-FISH and methods of BM analysis (CCA or BM-FISH). Correction of PB-FISH results to include only nonlymphoid or CD3(-) cells reduced the mean of differences and improved agreement between PB-FISH and CCA or BM-FISH methods. The best agreement was noted between CCA and PB-FISH on nonlymphoid cells. On the other hand, results of BM-FISH agreed well with those of PB-FISH on CD3(-) cells. These findings imply that PB-FISH on nonlymphoid or CD3(-) cells is reliable and may replace BM analysis for monitoring of response to treatment in CML patients.     

Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization

The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH). In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr). Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events. To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization. Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined. BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%). On cytogenetic preparations, the percentages of BCR-ABL- positive interphase cells ranged from 53% to 91%. Comparable efficiencies were achieved on blood smears. In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL.     

Effect of allogeneic stem cell transplantation on bone marrow angiogenesis in chronic myelogenous leukemia

Increased bone marrow angiogenesis is a poor prognostic marker in patients with chronic myelogenous leukemia (CML). Allogeneic stem cell transplantation (ASCT) can be curative for patients with CML. Studies in myeloma have shown persistent increased bone marrow microvessel density (MVD) after autologous transplantation. It is not clear if abnormal bone marrow angiogenesis persists following a curative intervention like allogeneic transplantation. We evaluated MVD from bone marrow samples obtained just prior to and at 3-5 months after ASCT in 24 patients with CML. The median MVD pre-transplant was 14 (4-37), with 11 patients having high-grade angiogenesis and 13 having low grade. The median post transplant MVD was 20 (range 5-36), with 12 patients having high-grade angiogenesis and 12 low grade. The median time between biopsies was 4 months (range 1-6 months). The microvessels in the post transplant bone marrow appeared morphologically different with striking dilatation and sinusoidal appearance compared to the pre-transplant marrow. However, there was no significant change in MVD following transplant (P=0.8, paired t-test). Abnormal bone marrow angiogenesis appears to persist in the bone marrow following ASCT for CML, at least in the short term.     

Ex vivo expansion and characterisation of CD34+ cells derived from chronic myeloid leukaemia bone marrow and peripheral blood, and from normal bone marrow and mobilised peripheral blood

Ex vivo culture of CD34+ has the potential to provide large numbers of cells for clinical use in autologous and allogeneic transplantation and for experimental research involving genetic manipulation. We evaluated the ex vivo expansion of CD34+ cells obtained from bone marrow (BM) and peripheral blood (PB) of untreated patients with chronic myeloid leukaemia (CML) in the chronic phase and compared these results with those obtained from BM from normal volunteers (NBM) and peripheral blood after mobilising chemotherapy from patients with non-haematological disorders (MPB). Selected CD34+ cells were stimulated with interleukin 1(beta), interleukin IL-3, interleukin IL-6 and stem cell factor. The proliferation observed in patients with CML was similar to that seen in normal donors. CD34+ cells derived from patients with CML are more differentiated than their normal counterparts, as shown by the coexpression of CD34 and CD33 antigens on day 0 (85.6% for CML-BM and 76.8% for CML-PB). The culture conditions allowed a significant expansion of granulocyte-macrophage colony-forming units (CFU-GM) from NBM (33-fold increase) and MPB (22-fold increase), in contrast with CML-derived BM and PB CD34+ cells (2.3-fold increase). These results indicate that the optimal time to harvest ex vivo expanded cells is dependent on a critical compromise between cell numbers and successful retention of their repopulating potential.     

Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure

SUMMARY. Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colonyforming cells, and AA progenitors fail to proliferate in longterm assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34⁺ and CD33⁺ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34⁺ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34⁺ cell reduction. A decrease in BM CD33⁺ cells was a common finding in MDS patients, but reduction in CD34⁺ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purged CD34⁺ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c-kit receptor expression on CD34⁺ cells, we found that the mitotically active CD34⁺ cells bearing the c-kit antigen were reduced in AA. With clinical improvement, CD34⁺ and CD33⁺ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34⁺ cells from recovered patents showed improved plating efficiency. In patients with aplastic anaemia, use of CD34 antigen as a phenotypic marker of progenitor cells may be helpful for the analysis of the early haemopoietic cell compartment and BM recovery.