love copy van cleef & arpels gold pendant How much it? from loersertydass's blog
Minimal residual disease cells in AML patients have an apoptosis
Correspondence: Dr GJ Schuurhuis, Department of Hematology, VU University Medical Center, BR240, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
TO THE EDITOR
Relapse in acute myeloid leukemia is common and is thought to arise from minimal residual disease (MRD) cells surviving chemotherapy.1 Survival of MRD cells has been attributed to drug resistance mechanisms. Defects in apoptosis pathways are probably contributing significantly to resistance to a variety of chemotherapeutic agents. Despite an emerging knowledge of the regulatory mechanisms of apoptosis, Bcl 2 still stands at the top of the list of parameters that contribute to chemotherapeutic resistance. In AML, it has been shown that Bcl 2 is of independent prognostic significance. High Bcl 2 predicts failure to achieve complete remission2 and shorter overall survival.3
In this study the expression profile of Bcl 2 and Bcl 2 related proteins in MRD cells was determined to examine whether chemotherapy induced upregulation of an apoptosis resistant protein profile or selection of subpopulations with a high anti apoptosis protein profile occurs during chemotherapeutic treatment.
In all, 61 AML patients were enrolled in this study after informed consent. Fresh bone marrow (BM) aspirates were obtained at diagnosis (n=61), after first cycle of induction chemotherapy (n=22), second cycle of induction (n=15) and after postinduction cycle (n=10) of chemotherapy, at follow up (n=13), and at relapse (n=9). The median age was 54 years, 29 male and 32 female subjects distributed over RAEB t (5 ), M0 (5 ), M1 (7 ), M2 (14 ), M3 (2 ), M4 (12 ), M5 (10 ), M6 (3 ), unknown (3 ) according to the FAB classification. In toal, 11 normal BM (nBM) from healthy donors were included.
To enable the study of apoptosis related protein expression in MRD cells, we relied upon an intracellular flow cytometry method.4,5 Briefly, this method consists of two parts; firstly, patient specific leukemia associated phenotypes (LAPs) are determined at diagnosis followed by an intracellular procedure to detect the Bcl 2 related proteins in these cells. The LAP can then be used to detect MRD cells in follow up BM samples. Figure 1a shows an example of an AML patient with a cross lineage LAP. Gating on the LAP+ cells enables quantification of the apoptosis related proteins of these cells (Figure replica van cleef jewelry 1c). The mean Bcl 2 expression was significantly lower in nBM CD34+ cells (Bcl 2: 6.62.5, P=0.001). Also, Bcl xL (2.20.8) and Mcl 1 (6.83.3) were lower and Bax was higher quatrefoil necklace van cleef replica (5.74.0), although these differences were not significant. All this suggests that the majority of AML cases have an increased anti apoptosis protein profile potentially offering the leukemic cells a survival advantage under therapy. This was tested with the same analysis procedure as at diagnosis, but was now performed on the MRD cells from patients in CR. Pgp activity analysis6 was performed in 17/51 MRD samples, showing deviant Pgp activity in MRD cells as compared to normal CD34+ cells confirming the malignant origin of the MRD cells. Figure 1b shows the MRD cells in a BM sample after the second cycle of induction chemotherapy. Surprisingly, the Bcl 2 expression (Figure 1d) of the MRD cells had decreased from 48 to 13. Of interest, this value now is near the range of nBM (range: 3.6 Figures 1e show the Bcl 2 value of all measured samples. In particular samples with high Bcl 2 levels (Figure 1e) but also with intermediate Bcl 2 levels (Figure 1f) and even with Bcl 2 levels within the normal range (Figure 1g) show decreases in expression. Overall, it can be concluded that MRD cells have significantly lower Bcl 2 levels after chemotherapeutic treatment (cycle 1: P=0.01, cycle 2: P=0.0008, cycle 3: P=0.04) and at follow up (FU1: P=0.01 and FU2: P=0.01) than at diagnosis. In all cases gating was performed on CD45low/SSClow cells (a) AML blasts at diagnosis with a CD34+CD7+ LAP. (b) BM sample after the second cycle of chemotherapy from the AML patient of (a) with MRD cells identified by the LAP (CD34+CD7+). MRD cells are 0.25% of all BM cells. Such cells are not present in control BM samples. Gating on the LAP+ cells enables quantification of Bcl 2 (c) at diagnosis and (d) after second cycle of induction chemotherapy. (e Summary of Bcl 2 levels of AML patients in CR after first, second, third cycle of chemotherapy at follow up (FU1 and FU2) and at relapse. of nBM CD34+ cells and (g) within the range found in nBM CD34+ cells (h) Bcl 2 levels of patients not in CR (NCR) at time of BM aspiration after first replica van cleef blue necklace and second cycle of chemotherapy and at relapse.
Full figure and legend (206K)
In contrast, Bcl 2 levels from refractory patients showed no change after first (P=0.64) or second cycle (P=0.79) (Figure 1h).
Despite the fact that the MRD cells in CR were apoptosis sensitive as compared to diagnosis, they were able to survive chemotherapy resulting in some cases in relapse. Bcl 2 expression at relapse had again increased to the level comparable to diagnosis (no difference with diagnosis; P=0.69) after the described decrease in an MRD situation.
Two other anti apoptosis proteins Bcl xL and Mcl 1 and the proapoptotic protein Bax were also studied (Table 1). In summary, although less prominent compared to Bcl 2, in responding patients Bcl xL and Mcl 1 significantly decreased from the second cycle onwards (Table 1a), while no change was observed in refractory patients (Table 1b). Relapse samples had similar protein levels as at diagnosis. On the other hand, Bax showed an increased expression compared to diagnosis, reaching significance after the first cycle of chemotherapy in responding patients. This shows that the prominent results found for the anti apoptosis proteins are not the results of aspecific treatment related cellular changes.
Overall, our data show that MRD blasts do not have a more apoptosis resistant profile. Instead, when a complete remission is achieved, in the majority of patients MRD cells are apoptosis sensitive compared to diagnosis and remain apoptosis sensitive throughout the remission period. Only when patients do not reach a CR or when patients relapse, the leukemic blasts retain their diagnosis apoptosis protein profile. These unexpected results clearly show that neither chemotherapy induced upregulation of an apoptosis resistant protein profile nor selection of subpopulations with a high anti apoptosis protein profile occurs. It does however suggest that external factors likely regulate expression of apoptosis related proteins. Either AML BM stromal cells or AML blasts themselves may account for this. Pertinent to this is the observation of Milojkovic et al (Milojkovic D, Buggins AG, Devereux S, Thomas NSB, Mufti GJ. Blood 2002; 100: 556a, 2184) that AML tumor supernatants can inhibit apoptosis of malignant hematopoietic cells. Combined with our observation this might indicate that AML cells at diagnosis, in follow up situations of refractory patients and at relapse produce apoptosis regulating factors that affect neighbouring cells, while in a CR BM due to the very limited number of AML cells, production is ineffective. Such cells might however be affected by the BM stroma: Konopleva et al7 found that a stromal cell line was able to increase Bcl 2 expression in blasts of 5/11 AML patients. It may then be hypothesized that intact BM stroma from AML patients may induce upregulation of anti apoptosis profiles in intact stroma, while such may be impossible in damaged stroma, the latter in fact resembling the actual situation in remission BM after chemotherapy.
Correspondence: Dr GJ Schuurhuis, Department of Hematology, VU University Medical Center, BR240, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
TO THE EDITOR
Relapse in acute myeloid leukemia is common and is thought to arise from minimal residual disease (MRD) cells surviving chemotherapy.1 Survival of MRD cells has been attributed to drug resistance mechanisms. Defects in apoptosis pathways are probably contributing significantly to resistance to a variety of chemotherapeutic agents. Despite an emerging knowledge of the regulatory mechanisms of apoptosis, Bcl 2 still stands at the top of the list of parameters that contribute to chemotherapeutic resistance. In AML, it has been shown that Bcl 2 is of independent prognostic significance. High Bcl 2 predicts failure to achieve complete remission2 and shorter overall survival.3
In this study the expression profile of Bcl 2 and Bcl 2 related proteins in MRD cells was determined to examine whether chemotherapy induced upregulation of an apoptosis resistant protein profile or selection of subpopulations with a high anti apoptosis protein profile occurs during chemotherapeutic treatment.
In all, 61 AML patients were enrolled in this study after informed consent. Fresh bone marrow (BM) aspirates were obtained at diagnosis (n=61), after first cycle of induction chemotherapy (n=22), second cycle of induction (n=15) and after postinduction cycle (n=10) of chemotherapy, at follow up (n=13), and at relapse (n=9). The median age was 54 years, 29 male and 32 female subjects distributed over RAEB t (5 ), M0 (5 ), M1 (7 ), M2 (14 ), M3 (2 ), M4 (12 ), M5 (10 ), M6 (3 ), unknown (3 ) according to the FAB classification. In toal, 11 normal BM (nBM) from healthy donors were included.
To enable the study of apoptosis related protein expression in MRD cells, we relied upon an intracellular flow cytometry method.4,5 Briefly, this method consists of two parts; firstly, patient specific leukemia associated phenotypes (LAPs) are determined at diagnosis followed by an intracellular procedure to detect the Bcl 2 related proteins in these cells. The LAP can then be used to detect MRD cells in follow up BM samples. Figure 1a shows an example of an AML patient with a cross lineage LAP. Gating on the LAP+ cells enables quantification of the apoptosis related proteins of these cells (Figure replica van cleef jewelry 1c). The mean Bcl 2 expression was significantly lower in nBM CD34+ cells (Bcl 2: 6.62.5, P=0.001). Also, Bcl xL (2.20.8) and Mcl 1 (6.83.3) were lower and Bax was higher quatrefoil necklace van cleef replica (5.74.0), although these differences were not significant. All this suggests that the majority of AML cases have an increased anti apoptosis protein profile potentially offering the leukemic cells a survival advantage under therapy. This was tested with the same analysis procedure as at diagnosis, but was now performed on the MRD cells from patients in CR. Pgp activity analysis6 was performed in 17/51 MRD samples, showing deviant Pgp activity in MRD cells as compared to normal CD34+ cells confirming the malignant origin of the MRD cells. Figure 1b shows the MRD cells in a BM sample after the second cycle of induction chemotherapy. Surprisingly, the Bcl 2 expression (Figure 1d) of the MRD cells had decreased from 48 to 13. Of interest, this value now is near the range of nBM (range: 3.6 Figures 1e show the Bcl 2 value of all measured samples. In particular samples with high Bcl 2 levels (Figure 1e) but also with intermediate Bcl 2 levels (Figure 1f) and even with Bcl 2 levels within the normal range (Figure 1g) show decreases in expression. Overall, it can be concluded that MRD cells have significantly lower Bcl 2 levels after chemotherapeutic treatment (cycle 1: P=0.01, cycle 2: P=0.0008, cycle 3: P=0.04) and at follow up (FU1: P=0.01 and FU2: P=0.01) than at diagnosis. In all cases gating was performed on CD45low/SSClow cells (a) AML blasts at diagnosis with a CD34+CD7+ LAP. (b) BM sample after the second cycle of chemotherapy from the AML patient of (a) with MRD cells identified by the LAP (CD34+CD7+). MRD cells are 0.25% of all BM cells. Such cells are not present in control BM samples. Gating on the LAP+ cells enables quantification of Bcl 2 (c) at diagnosis and (d) after second cycle of induction chemotherapy. (e Summary of Bcl 2 levels of AML patients in CR after first, second, third cycle of chemotherapy at follow up (FU1 and FU2) and at relapse. of nBM CD34+ cells and (g) within the range found in nBM CD34+ cells (h) Bcl 2 levels of patients not in CR (NCR) at time of BM aspiration after first replica van cleef blue necklace and second cycle of chemotherapy and at relapse.
Full figure and legend (206K)
In contrast, Bcl 2 levels from refractory patients showed no change after first (P=0.64) or second cycle (P=0.79) (Figure 1h).
Despite the fact that the MRD cells in CR were apoptosis sensitive as compared to diagnosis, they were able to survive chemotherapy resulting in some cases in relapse. Bcl 2 expression at relapse had again increased to the level comparable to diagnosis (no difference with diagnosis; P=0.69) after the described decrease in an MRD situation.
Two other anti apoptosis proteins Bcl xL and Mcl 1 and the proapoptotic protein Bax were also studied (Table 1). In summary, although less prominent compared to Bcl 2, in responding patients Bcl xL and Mcl 1 significantly decreased from the second cycle onwards (Table 1a), while no change was observed in refractory patients (Table 1b). Relapse samples had similar protein levels as at diagnosis. On the other hand, Bax showed an increased expression compared to diagnosis, reaching significance after the first cycle of chemotherapy in responding patients. This shows that the prominent results found for the anti apoptosis proteins are not the results of aspecific treatment related cellular changes.
Overall, our data show that MRD blasts do not have a more apoptosis resistant profile. Instead, when a complete remission is achieved, in the majority of patients MRD cells are apoptosis sensitive compared to diagnosis and remain apoptosis sensitive throughout the remission period. Only when patients do not reach a CR or when patients relapse, the leukemic blasts retain their diagnosis apoptosis protein profile. These unexpected results clearly show that neither chemotherapy induced upregulation of an apoptosis resistant protein profile nor selection of subpopulations with a high anti apoptosis protein profile occurs. It does however suggest that external factors likely regulate expression of apoptosis related proteins. Either AML BM stromal cells or AML blasts themselves may account for this. Pertinent to this is the observation of Milojkovic et al (Milojkovic D, Buggins AG, Devereux S, Thomas NSB, Mufti GJ. Blood 2002; 100: 556a, 2184) that AML tumor supernatants can inhibit apoptosis of malignant hematopoietic cells. Combined with our observation this might indicate that AML cells at diagnosis, in follow up situations of refractory patients and at relapse produce apoptosis regulating factors that affect neighbouring cells, while in a CR BM due to the very limited number of AML cells, production is ineffective. Such cells might however be affected by the BM stroma: Konopleva et al7 found that a stromal cell line was able to increase Bcl 2 expression in blasts of 5/11 AML patients. It may then be hypothesized that intact BM stroma from AML patients may induce upregulation of anti apoptosis profiles in intact stroma, while such may be impossible in damaged stroma, the latter in fact resembling the actual situation in remission BM after chemotherapy.
The Wall